靶向siRNA下调人结直肠癌Colo320细胞端粒长度与端粒酶逆转录酶基因表达的研究

目的:探讨发夹状shRNA封阻原癌基因(c-myc),抑制癌细胞增殖、生长的作用。方法:针对c-myc的构建发夹状shRNA的真核表达质粒,并转染人结直肠癌Colo320细胞。荧光定量RT-PCR检测c-myc及细胞端粒酶逆转录酶的mRNA的表达,Western-blot检测c-myc、端粒酶逆转录酶(hu-man telomerase reverse transeriptase,hTERT)蛋白表达水平。Southernblot检测端粒的长度,PCR-ELISE法检测端粒酶活性。3H-thymidine实验分析DNA合成和细胞增殖。结果:转染细胞增殖、生长皆受到抑制。同时c-myc和hTERT的mRNA和蛋白表达显著下降,端粒的长度明显缩短,端粒酶活性降低。结论:c-myc的shRNA对人结直肠癌Colo320细胞的生长增殖、端粒长度、端粒酶活性有特异性抑制作用,并呈剂量依赖关系。     

The evolving role of telomerase inhibitors in the treatment of cancer

SUMMARY

Telomerase is a ribonucleoprotein that maintains telomeres and is essential for cellular immortality and tumour growth. The differential expression of telomerase in cancer cells makes it an attractive therapeutic target. Anti-sense oligonucleotides directed against the RNA template of hTR and small molecules that can interact and stabilise the G-quadruplex represent promising therapeutic strategies. Human trials investigating the potential role of the catalytic subunit hTERT as a universal cancer vaccine have already commenced. Alternative lengthening of telomeres (ALT) and efficacy delay remain important limitations to anti-telomerase therapy.     

The evolving role of telomerase inhibitors in the treatment of cancer

Telomerase is a ribonucleoprotein that maintains telomeres and is essential for cellular immortality and tumour growth. The differential expression of telomerase in cancer cells makes it an attractive therapeutic target. Anti-sense oligonucleotides directed against the RNA template of hTR and small molecules that can interact and stabilise the G-quadruplex represent promising therapeutic strategies. Human trials investigating the potential role of the catalytic subunit hTERT as a universal cancer vaccine have already commenced. Alternative lengthening of telomeres (ALT) and efficacy delay remain important limitations to anti-telomerase therapy.     

Inorganic nanoparticle-based advanced cancer therapies: Promising combination strategies

Inorganic nanoparticles for drug delivery in cancer treatment offer many potential advantages because they can maximize therapeutic effect through targeting ligands while minimizing off-target side-effects through drug adsorption and infiltration. Although inorganic nanoparticles were introduced as drug carriers, they have emerged as having the capacity for combined therapeutic capabilities, including anticancer effects through cytotoxicity, suppression of oncogenes and cancer cell signaling pathway inhibition. The most promising advanced strategies for cancer therapy are as synergistic platforms for RNA interference (siRNA, miRNA, shRNA) and as synergistic drug delivery agents for the inhibition of cancer cell signaling pathways. The present work summarizes relevant current work, the promise of which is suggested by a projected compound annual growth rate of ～ 20% for drug delivery alone.     

反义hTERT体外诱导肺癌细胞凋亡的研究

目的研究反义hTERT基因体外诱导肺癌细胞凋亡。方法体外通过SuperFect(QIAGEN)转染正、反义hTERT真核表达载体至人肺腺癌细胞株GLC82,再经过G418及PCR筛选鉴定获得分别转入了正、反义hTERT载体的抗性克隆细胞GLC82s和GLC82as。然后采用MTT法、双层软琼脂克隆形成试验、流式细胞术观察和分析了反义hTERT对肺癌细胞体外生长增殖活力的影响及是否能诱导瘤细胞的凋亡;同时还运用实时荧光定量RTPCR技术及TRAP银染法对各组细胞内源性hTERTmRNA的表达情况及端粒酶的活性进行了检测。结果当各组细胞传至第25代后,与GLC82、GLC82s比较,GLC82as细胞的生长速度和集落形成能力明显地减慢和降低,同时伴有凋亡率的显著增加;与空白对照、正义hTERT组相比,反义hTERT能显著地降低GLC82细胞内源性hTERTmRNA的表达(P<0.01)和下调端粒酶活性。结论反义hTERT体外确实能明显地诱导肺癌细胞的凋亡及抑制瘤细胞的生长增殖能力。     

Harnessing telomerase in cancer therapeutics

Telomerase is an attractive target for anti-cancer therapeutics due to its requirement for cellular immortalization and expression in greater than 85% of human neoplasms. Though initially promising, strategies that inhibit telomerase with either small molecules or antisense oligonucleotides have a major limitation, namely the lag time required for telomere shortening before cellular effects are attained. As alternative approaches, immunotherapy and gene therapy have been tailored to exploit, rather than antagonize telomerase expression and/or activity. Immunotherapy requires the presence of the catalytic subunit of telomerase, hTERT, to elicit an immune response directed towards hTERT peptide-presenting cells. hTERT promoter-driven gene therapy and mutant telomerase RNA (hTR) gene therapy depend on the innate telomerase activity of cancer cells to drive the expression of pro-apoptotic genes and to synthesize mutated DNA sequences onto telomeres, respectively. In addition, we will discuss telomestatin, a G-quadruplex binding ligand that may exert anti-proliferative effects independently of telomere shortening. In this review, the progress, advantages, and limitations of these strategies in the ongoing effort to develop clinically relevant telomerase-based cancer therapeutics will be examined.     

汉黄芩素对人卵巢癌细胞株SKOV3裸鼠移植瘤生长及端粒酶活性的抑制作用

目的端粒酶在多种肿瘤细胞中高表达,可能是肿瘤分子靶向治疗的一个理想靶点。已有的研究表明,汉黄芩素体外具有抑制肿瘤细胞端粒酶活性和肿瘤细胞增殖的作用。该研究旨在评价汉黄芩素对人卵巢癌SKOV3细胞裸鼠移植瘤生长及端粒酶活性是否具有抑制作用。方法将移植人卵巢癌SKOV3细胞的裸鼠随机分为5组:汉黄芩素高剂量组(600mg·kg-1)、汉黄芩素低剂量组(300mg·kg-1)、空白对照组、顺铂治疗组(3mg·kg-1)和联合用药(顺铂+汉黄芩素)组,定期检测裸鼠的体重及肿瘤体积,裸鼠移植瘤组织分别提取DNA、RNA和蛋白质,用Southern印迹杂交法测定端粒长度、RT-PCR扩增观察端粒酶基因hTERT的表达、TRAP-PCR-银染法测定端粒酶活性。结果动物实验表明,汉黄芩素对SKOV3移植瘤有明显的抑瘤作用,高剂量组抑瘤率达56.67%(与对照组比较,P=0.002);低剂量组抑瘤率达38.10%(P=0.019),化疗组抑瘤率达50.83%(P=0.004),联合用药(顺铂+汉黄芩素)组的抑瘤率为66.9%,优于单独使用组(P=0.002);不同浓度汉黄芩素组肿瘤组织的端粒长度与空白对照组比较差异无显著性,汉黄芩素对瘤体端粒酶基因hTERT表达、端粒酶活性均有抑制作用,而且作用强度与剂量有关。结论汉黄芩素在体内具有抗肿瘤和抑制端粒酶活性的作用,抑制强度与剂量成正比,抑瘤率与顺铂合用有相加的作用。     

顺铂和依托泊苷下调肺癌细胞端粒酶量及端粒酶逆转录酶的表达

目的　观察肺癌化疗的一线药物顺铂 (DDP)和依托泊苷 (VP16 )对肺癌细胞端粒酶量和端粒酶逆转录酶基因表达和蛋白质表达的影响 ,从而进一步探讨这些药物在肺癌治疗中的作用机理。方法用不同浓度的化疗药物处理肺腺癌A5 49细胞 ,用噻唑蓝 (MTT)和流式细胞术观察化疗药物 (DDP和VP16 )对肺癌细胞的生长抑制和细胞凋亡情况 ,分别用端粒重复扩增 (TRAP)法、逆转录 聚合酶链反应 (RT PCR)法和Western印迹杂交检测处理后端粒酶量 ,以及其催化亚基端粒酶逆转录酶 (hTERT)基因和蛋白质的变化。结果　DDP和VP16明显抑制肺癌细胞生长 ,并诱导细胞凋亡 ,有剂量和时间依赖性 .DDP和VP16明显抑制端粒酶量和hTERT基因及蛋白表达 ,尤其以高剂量 (VP16 2 0 0mg·L- 1,DDP5 0mg·L- 1)和低剂量 (VP16 2 0mg·L- 1,DDP 5mg·L- 1)作用的d 5更为明显。结论　VP16和DDP可以抑制肺癌细胞端粒酶量 ,这种作用可能是通过抑制端粒酶的催化亚基hTERT的表达来实现的。端粒酶量测定可能有助于作为判断肺癌化疗时VP16和DDP疗效的观察指标     

Telomerase as drug and drug target for the treatment of thyroid cancer

Telomerase is known to be activated in almost all cancer cells and is quiescent in almost all normal cells. Therefore, it follows that therapeutic strategies directed against cancer would include the targeting of telomerase, as well as the use of telomerase. Several approaches have been used both in vitro and in vivo and include the following: 1) antisense; 2) immunotherapy directed against hTERT; and 3) the use of telomerase promoter to direct cytotoxic therapy. Herein we review these approaches and discuss their potential applicability against thyroid cancer.