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survivin 反义寡核苷酸逆转顺铂诱导的人肺腺癌细胞凋亡耐受作用[J]. 肿瘤防治研究, 2007, 34(08): 560-564. DOI: 10.3971/j.issn.1000-8578.540
引用本文: survivin 反义寡核苷酸逆转顺铂诱导的人肺腺癌细胞凋亡耐受作用[J]. 肿瘤防治研究, 2007, 34(08): 560-564. DOI: 10.3971/j.issn.1000-8578.540
Antisense Oligodeoxynucleotides Targeting survivin Gene Reverse Apoptotic Resistance Induced by Cisplatin in Human Lung Adenocarcinoma Cells[J]. Cancer Research on Prevention and Treatment, 2007, 34(08): 560-564. DOI: 10.3971/j.issn.1000-8578.540
Citation: Antisense Oligodeoxynucleotides Targeting survivin Gene Reverse Apoptotic Resistance Induced by Cisplatin in Human Lung Adenocarcinoma Cells[J]. Cancer Research on Prevention and Treatment, 2007, 34(08): 560-564. DOI: 10.3971/j.issn.1000-8578.540

survivin 反义寡核苷酸逆转顺铂诱导的人肺腺癌细胞凋亡耐受作用

Antisense Oligodeoxynucleotides Targeting survivin Gene Reverse Apoptotic Resistance Induced by Cisplatin in Human Lung Adenocarcinoma Cells

  • 摘要: 目的 探讨survivin反义寡核苷酸逆转人肺腺癌细胞对顺铂诱导的细胞凋亡耐受的效应。方法 1.常规体外培养A549/CDDP细胞,以脂质体包裹的survivin反义寡核苷酸(ASODN)体外转染细胞。2.采用二苯胺反应法(DPA)测定DNA片段化、激酶法检测Caspase-3酶活性及流式细胞术检测细胞凋亡率(AI),评价细胞凋亡程度。3.采用MTT法测定细胞存活率和生长抑制率,计算半效抑制浓度(IC50)及耐药倍数(RI)。结果 1.单用ASODN转染组DNA片段化比率、细胞凋亡率和Caspase-3相对活性分别增高至(43.55±6.07)%、(34.03±2.25)%和1.1298±0.2502,和各对照组相比较,均有显著性变化(P〈0.01);而ASODN和CDDP联用组上述凋亡指标增高更为明显,分别达(76.73±2.04)%、(65.85±5.47)%和1.6805±0.2758(P〈0.05)。2.转染组A549/CDDP细胞生长抑制显著,单用转染组和联合CDDP组生长抑制率分别增高达59.3%和83.7%(P〈0.05)。3.ASODN转染后CDDP对细胞IC50由(225.03±10.59)μmol/L减低至(158.84±4.256)μmol/L,其RI由11.9减为8.39。结论 survivin反义寡核苷酸体外转染可降低耐顺铂人肺腺癌细胞的凋亡阈值,从而较大程度上逆转其对顺铂诱导的细胞凋亡耐受。

     

    Abstract: Objective  The acquisition of resistance to apoptosis is a major reason behind t reatment failures.Ectopic expression of some apoptosis related factors, such as bcl-2 family and inhibitor apoptosis family proteins ( IAPs), can result in inhibition of apoptosis induced by chemotherapy agent s and thereby confer to drug resistance in malignancies. Regulation of those gene expressions may reverse the apoptosis resistance to chemical agents. In the present study, we t ransfect cisplatin-resistant human lung adeno-carcinoma A549/ CDDP cell lines to explore whether antisense oligodeoxynucleotides (ASODN) targeting survivin gene can reverse apoptotic resistance induced by cisplatin in human lung adenocarcinoma cells. Methods  A549/ CDDP cell lines were cultured routinely with RPMI-1640 medium. survivin ASODN mediated by cytofectin was t ransfected into the A549/ CDDP cells . The influence of ASODN t ransfection on apoptosis was determined by Diphenylamine assay(DPA), caspase-3 colorimet ric assay and flow cytometry. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was performed to detect the cell viability, half-maximum inhibitory concent ration ( IC50 ) and cisplatin resistant index ( R I) was thereby calculated, respectively. Results  In the A549/ CDDP cells t ransfected by survivin ASODN for 48hr, the percentage of DNA f ragmentation, apoptotic index and Caspase23 activity was enhanced to (43. 55 ±6. 07) %, (34. 03 ±2. 25) % and 1. 1298 ±0. 2502, respectively ( P < 0. 05), it was obviously significant, compared to the controls. While combination with ASODN and 10μmol/ L cisplatin caused far more obvious apoptotic alterations in the cells, of which the percentage of DNA fragmentation, AI and Caspase-3 activity was reached to (76. 73 ±2. 04) %, (65. 85 ±5. 47) % and 1. 6805 ±0. 2758 ( P < 0. 05), respectively, and even compared to single ASODN group, it was still significant ( P < 0. 05) . Transfected with survivin ASODN single and with combination of cisplatin for 48hrs, the rate of cell growth inhibition in the resistant cells was enhanced to 59. 3 % and 83. 7 %, respectively( P < 0. 05), while cell viability inversely had the lowest reduction. And the half-maximum inhibitory concent ration of cisplatin was reduced from 225. 03 ±10. 59μmol/ L to 158. 84 ±4. 256μmol/ L, while the resistant index was conversely reduced from 11. 9 to 8. 39. Non-sense oligodeoxynucleotides (NSODN) and liposome had no effect on the cells ( P> 0. 05) . Conclusion  Transfection of antisense oligodeoxynucleotides targeting survivin gene can reduce the apoptosis threshold, thereaf ter to great extent reverse apoptotic resistance induced by cisplatin in cisplatin-resistant human lung adenocarcinoma in vitro.

     

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