Ito Hideaki, Aoki Hiroshi, Kühnel Florian, Kondo Yasuko, Kubicka Stefan, Wirth Thomas, Iwado Eiji, Iwamaru Arifumi, Fujiwara Keishi, Hess Kenneth R, Lang Frederick F, Sawaya Raymond, Kondo Seiji
Department of Neurosurgery, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
J Natl Cancer Inst. 2006 May 3;98(9):625-36. doi: 10.1093/jnci/djj161.
Conditionally replicating adenoviruses (CRAds) can be engineered to replicate selectively in cancer cells and cause cancer-specific cell lysis; thus they are considered a promising cancer therapy.
To elucidate the mechanisms by which CRAds induce cancer-specific cell death, we infected normal human fibroblasts (MRC5, telomerase negative), human malignant glioma (U373-MG and U87-MG), human cervical cancer (HeLa), and human prostate cancer (PC3) cells (all telomerase positive) with CRAds regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad-GFP). Nonapoptotic autophagy was assessed in Ad-GFP- and hTERT-Ad-infected cells by examining cell morphology, the development of acidic vesicular organelles, and the conversion of microtubule-associated protein 1 light chain 3 from the cytoplasmic form to the autophagosome membrane form; signaling via mammalian target of rapamycin (mTOR), an autophagy-associated molecule, was monitored by western blot analysis. We also compared the growth of subcutaneous gliomas in nude mice that were treated by intratumoral injection with Ad-GFP or hTERT-Ad. Survival of athymic mice carrying intracranial gliomas treated by intratumoral injection with Ad-GFP or hTERT-Ad was compared by using the Kaplan-Meier method and the Cox-Mantel log-rank analysis. All statistical tests were two-sided.
hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas. hTERT-Ad-induced autophagy was associated with hTERT-Ad infection kinetics. The mTOR signaling pathway was suppressed in tumor cells and in subcutaneous gliomas treated with hTERT-Ad compared with GFP-Ad or no treatment as shown by reduced phosphorylation of mTOR's downstream target p70S6 kinase (p70S6K). hTERT-Ad treatment of mice (n = 7) slowed growth of subcutaneous gliomas (mean tumor volume = 39 mm3, 95% confidence interval [CI] = 23 to 54 mm3) compared with GFP-Ad treatment (n = 7) (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P < .001). Mice carrying intracranial tumors that were treated with three intratumoral injections of hTERT-Ad survived longer (53 days) than after treatment with GFP-Ad (29 days) (seven mice per group, difference = 24 days, 95% CI = 20 to 28 days; P < .001).
hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death.
条件性复制腺病毒（CRAds）可经改造使其在癌细胞中选择性复制并导致癌症特异性细胞裂解；因此，它们被认为是一种有前景的癌症治疗方法。
为阐明CRAds诱导癌症特异性细胞死亡的机制，我们用受人类端粒酶逆转录酶启动子调控的CRAds（hTERT-Ad）或对照非复制性腺病毒（Ad-GFP）感染正常人成纤维细胞（MRC5，端粒酶阴性）、人类恶性胶质瘤（U373-MG和U87-MG）、人类宫颈癌（HeLa）和人类前列腺癌（PC3）细胞（均为端粒酶阳性）。通过检查细胞形态、酸性囊泡细胞器的形成以及微管相关蛋白1轻链3从细胞质形式向自噬体膜形式的转化，评估Ad-GFP和hTERT-Ad感染细胞中的非凋亡自噬；通过蛋白质印迹分析监测自噬相关分子哺乳动物雷帕霉素靶蛋白（mTOR）的信号传导。我们还比较了经瘤内注射Ad-GFP或hTERT-Ad治疗的裸鼠皮下胶质瘤的生长情况。通过Kaplan-Meier法和Cox-Mantel对数秩检验比较经瘤内注射Ad-GFP或hTERT-Ad治疗的携带颅内胶质瘤的无胸腺小鼠的生存期。所有统计检验均为双侧检验。
hTERT-Ad在肿瘤细胞和皮下胶质瘤中诱导肿瘤特异性自噬性细胞死亡。hTERT-Ad诱导的自噬与hTERT-Ad感染动力学相关。与GFP-Ad或未治疗相比，hTERT-Ad处理的肿瘤细胞和皮下胶质瘤中mTOR信号通路受到抑制，表现为mTOR下游靶点p70S6激酶（p70S6K）磷酸化减少。与GFP-Ad治疗组（n = 7）相比，hTERT-Ad治疗组（n = 7）的小鼠在第7天时皮下胶质瘤生长减缓（平均肿瘤体积 = 39 mm3，95%置信区间[CI] = 23至54 mm3）（平均肿瘤体积 = 200 mm3，95% CI = 149至251 mm3）（体积差异 = 161 mm3，95% CI = 126至197 mm3；P <.001）。经三次瘤内注射hTERT-Ad治疗的携带颅内肿瘤的小鼠比经GFP-Ad治疗的小鼠存活时间更长（53天）（每组7只小鼠，差异 = 24天，95% CI = 20至28天；P <.001）。
hTERT-Ad可能通过诱导自噬性细胞死亡杀死端粒酶阳性癌细胞。
