共查询到20条相似文献,搜索用时 31 毫秒
1.
Miura Y Ohnami S Yoshida K Ohashi M Nakano M Ohnami S Fukuhara M Yanagi K Matsushita A Uchida E Asaka M Yoshida T Aoki K 《Cancer letters》2005,218(1):53-62
We examined the antitumor effect and safety of the adenovirus-mediated expression of antisense K-ras RNA in two peritoneal dissemination models of pancreatic cancer. First, we found that the infection of an adenovirus vector expressing antisense human K-ras RNA (AxCA-AS) induced significant apoptosis in vitro in human pancreatic cancer cells with K-ras mutation. Second, the intraperitoneal (ip) injection of AxCA-AS effectively suppressed the growth of human pancreatic cancer cells in the peritoneal cavity of nude mice. Third, in the hamster syngeneic peritoneal dissemination model, the ip injection of an adenovirus expressing antisense hamster K-ras RNA significantly suppressed the peritoneal growth of hamster pancreatic cancer cells, and no significant systemic toxicity was observed in the treated hamsters. This study suggests a feasibility of the development of a therapeutic strategy against pancreatic cancer based on the adenovirus-mediated transduction of an antisense K-ras construct. 相似文献
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Despite successes in animal models, cancer gene therapy with small interfering RNAs (siRNA) is hindered by the lack of an optimal delivery platform. We examined the applicability of the replication-competent, oncolytic adenovirus, ONYX-411, to deliver a mutant K-ras siRNA transgene to human cancer cells. Proof-of-principle studies showed an additive tumor growth-inhibitory response through siRNA-mediated K-ras knockdown and ONYX-411-mediated cancer cell lysis. A novel construct, termed Internavec (for interfering RNA vector), was generated by cloning a K-ras(v12)-specific siRNA(ras-4) hairpin construct under the control of the human H1 promoter into the deleted E3b region of ONYX-411. Internavec acquired an increase in potency of approximately 10-fold in human cancer cells expressing the relevant K-ras(v12) mutation (H79, H441, and SW480), as defined by a reduction in the effective dose needed to achieve 50% growth inhibition (ED(50)). Internavec remained attenuated in nonmalignant epithelial cells. Daily intratumoral injections of Internavec (five daily injections of 1 x 10(8) plaque-forming units) significantly reduced the growth of s.c. H79 pancreatic cancer xenografts in nu/nu mice by 85.5%, including complete growth suppression in three of five mice. Parental ONYX-411 or ONYX-411-siRNA(GFP) was markedly less effective (47.8% growth reduction, P = 0.03; and 44.1% growth reduction, P = 0.03, respectively). siRNA(ras) transgene activity contributed to cell cycle blockage, increased apoptosis, and marked down-regulation of Ras signaling-related gene expression (AKT2, GSK3 beta, E2F2, and MAP4K5). These findings indicate that Internavec can generate a two-pronged attack on tumor cells through oncogene knockdown and viral oncolysis, resulting in a significantly enhanced antitumor outcome. 相似文献
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Jones-Bolin SE; Johansson E; Palmisano WA; Anderson MW; Wiest JS; Belinsky SA 《Carcinogenesis》1998,19(8):1503-1508
Differences in tumor formation among inbred mouse strains with high (A/J)
and low (C3H) susceptibility for lung cancer have been linked to a
repetitive element within the second intron of the K-ras gene. The purpose
of this investigation was to determine whether differences within the K-ras
gene promoter region or the intron 2 polymorphism affect K-ras gene
expression in lung tumors and target alveolar type II cells isolated from
A/J and C3H mice. Ribonuclease protection assays were performed using RNA
isolated from 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK)-induced
lung tumors from each mouse strain and alveolar type II cells isolated from
A/J and C3H mice. An 838 bp fragment of the murine K-ras gene promoter
region was amplified by PCR, cloned and sequenced from both mouse strains.
Promoter regions from both mouse strains were inserted into a luciferase
reporter gene vector, with and without the second intron polymorphism, and
transfected into sensitive, intermediate and resistant lung tumor cell
lines. No significant differences in K-ras gene promoter activity was found
between the two strains using these specific reporter gene constructs.
Consistent with these results, levels of K-ras expression did not differ
between alveolar type II cells, whole lung or tumors induced by NNK in A/J
or C3H mice. Moreover, in lung tumor cell lines derived from mice with
differing susceptibility for lung cancer, K-ras expression did not
correlate with the growth rate of tumors induced in nude mice from these
cell lines. These results indicate that factors involved in modulating the
rapid clonal expansion of the mutated K-ras allele from A/J mice are not
directly linked to expression of this gene. Other genetic changes or losses
in conjunction with hypothesized modifier loci, such as the Par1 locus,
must play a significant role in establishing the phenotypic strain
differences for lung tumor formation.
相似文献
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Knockdown of mutant K-ras expression by adenovirus-mediated siRNA inhibits the in vitro and in vivo growth of lung cancer cells 总被引:1,自引:0,他引:1
The ras mutation, which is observed in 20-30% of human nonsmall cell lung cancers (NSCLCs), is one of common genetic alterations and has been proposed to be a prognostic factor in lung cancer. Oncogene ras appears to be essential for tumor progression and maintenance. Several therapeutic agents have been developed to inhibit ras, such as FTIs and antisense oligonucleotides. A new tool for blocking oncogenes in cancer cells has emerged with the discovery that RNA interference can specifically silence expression of endogenous human genes. In the current study, we used small interfering RNA (siRNA) directed against mutant K-ras to determine the anti-tumor effects of decreasing the levels of this protein in lung cancer cell lines. Adenovirus-mediated siRNA (AdH1/siK-ras(V12)) against K-ras(V12) markedly decreased K-ras(V12) gene expression and inhibited cellular proliferation of NSCLC H441 cells that express the relevant mutation (K-ras codon 12 GGT --> GTT), but produced minimal growth inhibition on NSCLC H1650 cells that lack the relevant mutation. Pretreatment with AdH1/siK-ras(V12) completely abrogated subcutaneous engraftment of H441 cells, as compared with a 100% tumor take in animals that received control vector-treated tumor cells. The in vivo effect of AdH1/siK-ras(V12) treatment was further examined by intratumoral injections after tumor induction. Pre-existing tumor growth was reduced by 45% by a single intratumoral injection. Three or five repeat injections resulted in complete tumor regression in eight of ten nude mice. Further, 23.12% of AdH1/siK-ras(V12) treated H441 cells underwent apoptosis, as compared with 6.13%, and 8.27% in untreated and control vector-treated cells, respectively. These results indicate that adenovirus-mediated siRNA can specifically and efficiently target factors whose expression is altered in malignancy and may have the potential as a therapeutic modality to treat human lung cancer. 相似文献
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The prognosis of pancreatic carcinoma is poor due to the difficulty in early diagnosis, insensitivity to routine therapies and limited understanding of its pathological mechanisms. Gene therapy is now becoming an important strategy for the treatment of pancreatic carcinoma, which includes antisense gene therapy. In this study, we investigated the effect of an antisense oligonucleotide specific to point mutated K-ras on the apoptosis of human pancreatic carcinoma cells in vitro. Human pancreatic carcinoma PC-2 cells were transfected with an antisense oligonucleotide specific to a K-ras point mutation by liposomes. The effect of the antisense oligonucleotide on the apoptosis of PC-2 cells was studied using flow cytometry, TUNEL, and phase contrast microscopy. An apoptotic peak was observed in the experimental group, and most cells were arrested at the G1 phase with few cells at the S phase. The numbers of apoptotic cells in the experimental group increased as indicated by TUNEL and phase contrast microscopy. An antisense oligonucleotide specific to a K-ras point mutation promotes apoptosis in PC-2 cells in vitro perhaps by inhibition of ras gene expression. 相似文献
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Allele-specific detection of K-ras oncogene expression in human non-small-cell lung carcinomas 总被引:2,自引:0,他引:2
R J Slebos G G Habets S G Evers W J Mooi S Rodenhuis 《International journal of cancer. Journal international du cancer》1991,48(1):51-56
Point mutations in codon 12 of the K-ras oncogene are frequent in human lung adenocarcinomas. To study the expression of the K-ras gene in these tumors we have developed a mRNA detection technique based on the polymerase chain reaction (PCR). By this technique, K-ras expression can be detected semi-quantitatively in samples of less than 100 ng total RNA. Hybridization of the amplified cDNA sequences with mutation-specific oligonucleotides allows separate quantification of the expression of normal and point-mutated alleles in a single sample. RNA samples from 24 human non-small-cell lung carcinomas (NSCLC), from 2 lung metastases of colonic adenocarcinomas, from 3 human lung adenocarcinoma cell lines, and from normal lung tissue were analyzed. In most tumors, expression of K-ras was detected at levels equal to or several times higher than those found in normal lung tissue. A lung metastasis from a colon adenocarcinoma, known to contain an amplified K-ras gene, highly over-expressed the K-ras gene. In those tumors in which the K-ras oncogene was activated by a point mutation, both alleles of the gene were expressed. Our results show that a high over-expression of K-ras is a rare event in human lung carcinomas, but that a certain degree of over-expression of the mutated allele can be demonstrated in tumors with an activated K-ras gene. With the technique we describe here, adequate estimation of the expression of specific genes in minimal amounts of tumor cells becomes possible. 相似文献
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XIAP and survivin as therapeutic targets for radiation sensitization in preclinical models of lung cancer 总被引:20,自引:0,他引:20
Survivin and XIAP are members of inhibitors of apoptosis (IAPs) family. They are upregulated in various malignancies. Inactivation of these molecules has resulted in chemosensitization. The purpose of this study was to determine whether inhibition of survivin, XIAP, or both enhances radiotherapy in a lung cancer model. Transient transfection of H460 cells with antisense oligonucleotides (ASOs) against either molecule has specifically reduced their expression, by Western analysis. Results from 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and clonogenic assays suggest that inhibition of survivin or XIAP greatly decreased cell survival following irradiation. A significantly increased number of apoptotic cells were detected when H460 cells were treated with either antisurvivin, anti-XIAP or both ASOs (P=0.03, 0.0003 and 0.01, respectively) plus irradiation. H460 xenografts that were treated with ASOs plus radiotherapy demonstrated growth delay beyond 15 days. Growth delay in the groups of combined treatment was greater than that in other groups. However, treatment with ASOs alone did not affect tumor growth delay in mice, but decreased the survival of H460 cells in culture. Antisense treatment did not cause any mortality or weight loss during the 32 days of study. These data suggest that inhibition of survivin or XIAP radiosensitizes H460 lung cancer cells by upregulating apoptosis and downregulating cell survival. Combination of radiotherapy and inhibition of survivin and XIAP through the antisense approach results in improved tumor control by radiotherapy in a mouse model of lung cancer. 相似文献
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J A Roth T Mukhopadhyay M A Tainsky K Fang A G Casson P M Schneider 《Journal of the National Cancer Institute. Monographs》1992,(13):15-21
We describe studies defining several molecular events in human non-small-cell lung cancer (NSCLC). These include increased growth factor and growth factor receptor expression and oncogene alterations. The epidermal growth factor receptor (EGFR) and erbB2 are expressed by NSCLC cells. Transforming growth factor-alpha (TGF-alpha) is produced by NSCLC and may mediate autocrine growth stimulation. Specific inhibition of K-ras oncogene expression by an antisense K-ras construct reduces the growth rate and tumorigenicity of NSCLC cells. Studies with antisense p53 in NSCLC with a homozygous p53 mutation suggest that the presence of the mutant form contributes to the transformed state. 相似文献
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p53反义RNA对肠癌细胞恶性表型的抑制作用 总被引:8,自引:0,他引:8
研究p53反义RNA对大肠癌细胞中突变型p53致癌性的抑制效应,为肿瘤基因治疗提供新思路。方法2.1kb的人p53全长cDNA反向插入哺乳动物表达载体pREP9,得到p53反义RNA表达载体pREP9-p53(AS),并将其导入人肠癌细胞株SW1116(内源性突变型p53),采用MTT和FCM方法测定pREP9-p53(AS)表达的p53反义RNA对SW1116细胞生长的影响。结果带有pREP9-p53(AS)的SW1116细胞,与对照组SW1116细胞和带pREP9空载体的SW1116细胞相比,由于p53反义RNA的表达,其增殖速度显著下降,FCM的结果也证明带pREP9-p53(AS)的细胞部分受阻于G0/1期,而带pREP9空载体的细胞则无明显变化。结论p53反义RNA可以有效地抑制大肠癌细胞中突变型p53的致癌性,可用于实验性肿瘤基因治疗研究 相似文献
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目的:探讨TBX4基因表达对肺癌H460细胞增殖和侵袭转移能力等生物学行为的影响,并初步探讨其可能的分子机制。方法:采用RNA干扰的方法降低肺癌H460细胞TBX4的基因表达,采用磺酰罗丹明B(SRB)法检测细胞增殖能力;Transwell小室法检测肿瘤细胞迁移和侵袭能力的变化;蛋白质印迹法检查干扰前后TBX4及细胞增殖和转移相关基因CDKN2A、MTA2和MTSS1的表达。结果:RNA干扰TBX4后H460细胞增殖活性显著降低;RNA干扰TBX4组迁移和侵袭细胞数较对照组减少,但差异无统计学意义;TBX4表达下调后抑癌基因CDKN2A表达上调,但转移相关基因MTA2和MTSS1表达无明显变化。结论:TBX4对肿瘤细胞的恶性生物学行为可能具有促进作用,其机制有待进一步研究。 相似文献
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目的:研究慢病毒介导的β4整合素(integrinβ4,ITGB4) shRNA对人非小细胞肺癌(non-small cell lung cancer,NSCLC)裸鼠皮下移植瘤生长的抑制作用.方法:采用实时荧光定量-PCR (real-time fluorogenic quantitative-PCR,RFQ-PCR)和蛋白质印迹法检测ITGB4在不同人NSCLC细胞中的表达水平;慢病毒介导ITGB4 shRNA抑制NSCLC H460SM细胞中ITGB4的表达,再用RFQ-PCR和蛋白质印迹法检测ITGB4的表达水平,评价基因沉默效率;裸鼠随机分为3组,皮下分别接种H460SM-NS细胞(对照组)、稳定表达ITGB4 shRNA的H460SM-68和H460SM-71细胞.每隔1d测量裸鼠体质量和肿瘤大小,比较各组裸鼠肿瘤生长情况.处死裸鼠,剥离肿瘤,测量肿瘤大小和质量.结果:ITGB4在大多数人NSCLC细胞中均有表达,其中大细胞肺癌H460SM和NCI-H460细胞中ITGB4表达量明显高于NCI-H661细胞(P<0.01),H460SM细胞中ITGB4表达水平明显高于亲本NCI-H460细胞(P<0.01):ITGB4 shRNA转染组细胞中ITGB4的表达水平明显低于阴性对照组细胞(P<0.01); ITGB4 shRNA转染组皮下移植瘤生长速度明显低于阴性对照组(P<0.01).结论:ITGB4表达可能与人NSCLC细胞的致瘤、侵袭、转移的表型有关;抑制ITGB4的表达,可以抑制人NSCLC细胞H460SM裸鼠皮下移植瘤的生长. 相似文献
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Clusterin as a therapeutic target for radiation sensitization in a lung cancer model 总被引:4,自引:0,他引:4
Cao C Shinohara ET Li H Niermann KJ Kim KW Sekhar KR Gleave M Freeman M Lu B 《International journal of radiation oncology, biology, physics》2005,63(4):1228-1236
PURPOSE: Clusterin plays important roles in cell survival and death. Inactivation of clusterin enhances the therapeutic efficacy of chemotherapy in lung cancer models. The purpose of this study was to determine whether inhibition of clusterin by an antisense-based investigative drug enhances radiation sensitization in a lung cancer model. METHODS AND MATERIALS: Cells were transfected with an antisense oligonucleotide (ASO) against clusterin (OGX-011). Apoptosis was determined by 7-aminoactinomycin D staining. Cell survival was examined by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) and clonogenic assay. Xenograft model was used to demonstrate tumor growth and tumor blood flow. RESULTS: OGX-011 specifically attenuated the expression of secreted clusterin (prosurvival), with no apparent effect on the expression of nuclear clusterin (proapoptotic). Apoptosis was significantly increased when H460 lung cancer cells were treated with OGX-011 plus radiation. Inhibition of clusterin followed by radiation greatly decreased cell survival. H460 xenografts that were treated with OGX-011 plus radiotherapy demonstrated growth delay beyond 17 days. Doppler studies showed that tumor blood flow was compromised when mice bearing H460 xenografts were treated with OGX-011 and radiation. CONCLUSION: A combination of radiotherapy and OGX-011 improved control of tumor growth and vascular regression in the H460 lung cancer model. 相似文献
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MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer 总被引:1,自引:0,他引:1
Chattopadhyay S Machado-Pinilla R Manguan-García C Belda-Iniesta C Moratilla C Cejas P Fresno-Vara JA de Castro-Carpeño J Casado E Nistal M Gonzalez-Barón M Perona R 《Oncogene》2006,25(23):3335-3345
Non-small-cell lung cancer (NSCLC) represents the most frequent and therapy-refractive sub-class of lung cancer. Improving apoptosis induction in NSCLC represents a logical way forward in treating this tumor. Cisplatin, a commonly used therapeutic agent in NSCLC, induces activation of N-terminal-c-Jun kinase (JNK) that, in turn, mediates induction of apoptosis. In analysing surgical tissue samples of NSCLC, we found that expression of MKP1/CL100, a negative regulator of JNK, showed a strong nuclear staining for tumor cells, whereas, in normal bronchial epithelia, MKP1 was localized in the cytoplasm as well as in nuclei. In the NSCLC-derived cell lines H-460 and H-23, we found that MKP1 was constitutively expressed. Expressing a small-interfering RNA (siRNA) vector for MKP1 in H-460 cells resulted in a more efficient activation by cisplatin of JNK and p38 than in the parental cells, and this correlated with a 10-fold increase in sensitivity to cisplatin. A similar response was also observed in H-460 and H-23 cells when treated with the MKP1 expression inhibitor RO-31-8220. Moreover, expression of a siRNA-MKP2, an MKP1-related phosphatase, had no effect on H-460 cell viability response to cisplatin. Tumors induced by H-460 cells expressing MKP1 siRNA grew slower in nu(-)/nu(-) mice and showed more susceptibility to cisplatin than parental cells, and resulted in an impaired growth of the tumor in mice. On the other hand, overexpression of MKP1 in the H-1299 NSCLC-derived cell line resulted in further resistance to cisplatin. Overall, the results showed that inhibition of MKP1 expression contributes to a slow down in cell growth in mice and an increase of cisplatin-induced cell death in NSCLC. As such, MKP1 can be an attractive target in sensitizing cells to cisplatin to increase the effectiveness of the drug in treating NSCLC. 相似文献
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目的 探讨针对人RON基因跨膜区段(RONm)反义核酸对肺癌细胞系A549活性和细胞增殖的抑制作用,研究以RON基因为靶的肺癌基因治疗的可能性。方法 将人RONm cDNA反向插入到真核表达载体pcDNA3、1中,转染肺癌细胞A549,利用ELISA检测细胞模型RON基因的表达水平,同时利用MTT方法监测细胞生物学活性的变化。结果 转染后的肺癌细胞RON基因表达量和细胞活性明显降低,并出现明显的细胞凋亡。结论 RONm反义核酸明显抑制RON基因的表达,同时对肺癌细胞的生长有抑制作用,促进细胞凋亡。 相似文献
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目的:研究抑制clusterin的表达对肺癌H460细胞放射敏感性的影响。方法:用脂质体转染的方法将抗clusterin反义核酸转染肺癌H460细胞,以^137Cs作为放射源对肺癌细胞进行放射干预。用流式细胞仪检测抗clusterin反义核酸治疗和放射干预对肺癌细胞凋亡的影响,用MTT法和克隆形成分析方法检测抗clusterin反义核酸治疗和放射干预对肺癌细胞增殖的影响。结果:抗clusterin反义核酸特异性抑制了分泌型clusterin的表达,而对核型clusterin的表达无明显作用。单纯放射干预或单纯反义核酸转染将凋亡细胞数提高了10%,反义核酸治疗联合放射干预使肺癌H460细胞的凋亡细胞数增加了25%,和单纯放疗或单纯反义核酸转染组比较有显著性差异(P〈0.01)。MTT和体外克隆计数分析显示抗clusterin反义核酸治疗联合放疗干预显著抑制了肺癌H460细胞的增殖(P〈0.01)。结论:体外实验提示抗clusterin反义核酸治疗可以提高肺癌细胞对放射的敏感性。 相似文献
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目的:探讨慢病毒介导α6整合素(integrinα6,ITGA6)shRNA对人非小细胞肺癌(non-small cell lung cancer,NSCLC)H460SM细胞生长和侵袭的影响。方法:实时定量PCR和Western blotting检测ITGA6在9种NSCLC细胞中的表达。慢病毒介导ITGA6 shRNA感染H460SM细胞后,实时定量PCR和Wesern blotting检测H460SM中ITGA6的表达,显微镜下观察H460SM细胞形态的变化,MTS法检测细胞增殖,软琼脂实验检测H460SM细胞锚定非依赖性生长,流式细胞术检测细胞凋亡和细胞周期,迁移和侵袭实验检测H460SM细胞体外迁移和侵袭能力。结果:ITGA6在7种人NSCLC细胞中均有表达,H460SM细胞中ITGA6表达水平明显高于亲本H460细胞[(8.75±0.09)vs(5.78±0.26),P<0.01]。慢病毒介导ITGA6shRNA稳定感染H460SM细胞后的H460SM-75、H460SM-76细胞中,ITGA6表达水平明显低于阴性对照组H460SM-NS细胞[(0.11±0.04)、(0.22±0.04)vs(1.00±0.01),P<0.01]。H460SM-75、H460SM-76细胞增殖活性与H460SM-NS细胞无差异(P>0.05),且凋亡率、细胞增殖指数、G0/G1期细胞的比例也无差异(P>0.05)。H460SM-75、H460SM-76细胞的克隆形成率明显低于H460SM-NS细胞[(18.87±1.47)%、(18.85±1.11)%vs(20.81±1.38)%,P<0.05],迁移率[(43.92±0.41)%、(24.10±0.33)%vs(100.00±0.50)%,P<0.01]和侵袭率[(7.04±2.96)%、(4.68±0.27)%vs(100.00±6.74)%,P<0.01]也明显低于H460SM-NS细胞。结论:抑制ITGA6的表达可以抑制NSCLC细胞锚定非依赖性生长、迁移和侵袭,但不影响NSCLC细胞增殖和凋亡。 相似文献