Conditioned medium from Ad-IFN-α-infected bladder cancer and normal urothelial cells is cytotoxic to cancer cells but not normal cells: further evidence for a strong bystander effect
Conditioned medium from Ad-IFN-α-infected bladder cancer and normal urothelial cells is cytotoxic to cancer cells but not normal cells: further evidence for a strong bystander effect
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DOI：
10.1038/cgt.2008.53
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发表时间：
2008-12-01
影响因子：
6.4
通讯作者：
Benedict, W. F.
中科院分区：
文献类型：
--
作者：
Zhang, X.;Dong, L.;Benedict, W. F.
我们之前曾报道过，当用腺病毒介导的干扰素-α (Ad-IFN-α) 治疗时，在对高浓度干扰素-α 蛋白 (内含子 A) 产生耐药性的癌细胞中观察到旁观者效应。现在，我们使用从 Ad-IFN-α 感染的癌症和正常尿路上皮细胞收集的条件培养基（CM）为这种旁观者效应提供了进一步的证据。从 UC-9 和 KU7 膀胱癌细胞以及正常尿路上皮细胞中收集的 CM 在转染 Ad-IFN 后，在添加到培养物中的各种癌细胞类型中时会产生细胞死亡，但不会添加到正常尿路上皮细胞中。可以将CM过滤、冷冻和解冻，并稀释为至少一份Ad-IFN CM与五份新鲜对照培养基，并且稀释的CM仍然显示出与100%浓度的Ad-IFN CM相似的细胞毒性。通过流式细胞术、MTT 测定以及相差显微镜观察到这种细胞毒性，并且无论在初始 Ad-IFN 治疗后 48、72 还是 96 小时收集 CM，都可以看到显着的亚 G1 群体。此外，暴露于 65°C 30 分钟可部分灭活 CM，置于 92°C 3 分钟可完全灭活 CM，而内含子 A 在相同条件下未灭活。重要的是，尽管 Ad-IFN 转染的直接作用在 Ad-IFN 处理的细胞中发生了显着的 caspase 8 和 caspase 9 裂解，但 Ad-IFN CM 没有产生 caspase 8 和 caspase 9 的激活，表明旁观者因素产生的细胞死亡机制与 Ad-IFN 的直接作用不同。这种旁观者效应反过来可能对目前正在进行的浅表性膀胱癌 Ad-IFN 临床试验的疗效发挥重要作用。
We have reported earlier that a bystander effect is seen in cancer cells that are resistant to high concentrations of the interferon-alpha protein (Intron A) when treated with adenoviral-mediated interferon-alpha (Ad-IFN-alpha). We now provide further evidence for this bystander effect using conditioned medium (CM) collected from Ad-IFN-alpha-infected cancer and normal urothelial cells. The CMs collected from UC-9 and KU7 bladder cancer cells as well as normal urothelial cells following transfection with Ad-IFN produce cell death when added to various cancer cell types in culture but not to normal urothelial cells. The CM could be filtered, frozen and thawed, and diluted to at least one part Ad-IFN CM to five parts fresh control medium and the diluted CM still shows a similar cytotoxicity as a 100% concentration of Ad-IFN CM. This cytotoxicity was observed by both flow cytometry and MTT assays as well as by phase microscopy, and a significant sub-G1 population was seen whether the CM was collected 48, 72 or 96 h after initial Ad-IFN treatment. In addition, the CM could be partially inactivated by exposure to 65 degrees C for 30 min and totally inactivated by placement at 92 degrees C for 3 min, whereas Intron A was not inactivated under the same conditions. Importantly, although significant caspase 8 and caspase 9 cleavage occurred in Ad-IFN-treated cells as a direct effect of Ad-IFN transfection, the Ad-IFN CM produced no activation of caspase 8 and caspase 9, indicating that a different mechanism of cell death was produced by the bystander factor(s) than the direct effect of Ad-IFN. This bystander effect in turn may play an important role in the efficacy of the current Ad-IFN clinical trial for superficial bladder cancer now underway.