Department of Microbiology, Faculty of Basic Science, Qom Branch, Islamic Azad University, Qom, Iran.
Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
BMC Cancer. 2023 Jun 6;23(1):519. doi: 10.1186/s12885-023-11021-y.
Melanoma differentiation-associated gene 7 (Mda-7) encodes IL-24, which can induce apoptosis in cancer cells. A novel gene therapy approach to treat deadly brain tumors, recombinant mda-7 adenovirus (Ad/mda-7) efficiently kills glioma cells. In this study, we investigated the factors affecting cell survival and apoptosis and autophagy mechanisms that destroy glioma cells by Ad/IL-24.
Human glioblastoma U87 cell line was exposed to a multiplicity of infections of Ad/IL-24. Antitumor activities of Ad/IL-24 were assessed by cell proliferation (MTT) and lactate dehydrogenase (LDH) release analysis. Using flow cytometry, cell cycle arrest and apoptosis were investigated. Using the ELISA method, the tumor necrosis factor (TNF-α) level was determined as an apoptosis-promoting factor and Survivin level as an anti-apoptotic factor. The expression levels of TNF-related apoptosis inducing ligand(TRAIL) and P38 MAPK genes were assessed by the Reverse transcription-quantitative polymerase chain reaction(RT‑qPCR) method. The expression levels of caspase-3 and protein light chain 3-II (LC3-II) proteins were analyzed by flow cytometry as intervening factors in the processes of apoptosis and autophagy in the cell death signaling pathway, respectively.
The present findings demonstrated that transduction of IL-24 inhibited cell proliferation and induced cell cycle arrest and cell apoptosis in glioblastoma. Compared with cells of the control groups, Ad/IL24-infected U87 cells exhibited significantly increased elevated caspase-3, and TNF-α levels, while the survivin expression was decreased. TRAIL was shown to be upregulated in tumor cells after Ad/IL-24 infection and studies of the apoptotic cascade regulators indicate that Ad/IL-24 could further enhance the activation of apoptosis through the TNF family of death receptors. In the current study, we demonstrate that P38 MAPK is significantly activated by IL-24 expression. In addition, the overexpression of mda-7/IL-24 in GBM cells induced autophagy, which was triggered by the upregulation of LC3-II.
Our study demonstrates the antitumor effect of IL-24 on glioblastoma and may be a promising therapeutic approach for GBM cancer gene therapy.
黑色素瘤分化相关基因 7（Mda-7）编码白细胞介素 24（IL-24），可诱导癌细胞凋亡。一种新型的基因治疗方法来治疗致命的脑肿瘤，重组 mda-7 腺病毒（Ad/mda-7）可有效杀死神经胶质瘤细胞。在这项研究中，我们研究了影响细胞存活和凋亡的因素，以及通过 Ad/IL-24 破坏神经胶质瘤细胞的自噬机制。
人神经胶质瘤 U87 细胞系暴露于多倍感染的 Ad/IL-24。通过细胞增殖（MTT）和乳酸脱氢酶（LDH）释放分析评估 Ad/IL-24 的抗肿瘤活性。通过流式细胞术研究细胞周期停滞和凋亡。通过 ELISA 法测定肿瘤坏死因子（TNF-α）水平作为促进凋亡的因子和 Survivin 水平作为抗凋亡因子。通过逆转录-定量聚合酶链反应（RT-qPCR）法评估 TNF 相关凋亡诱导配体（TRAIL）和 P38 MAPK 基因的表达水平。通过流式细胞术分析 caspase-3 和蛋白轻链 3-II（LC3-II）蛋白的表达水平，分别作为细胞死亡信号通路中凋亡和自噬过程的干预因素。
本研究结果表明，IL-24 的转导抑制了神经胶质瘤的细胞增殖，并诱导了细胞周期停滞和细胞凋亡。与对照组细胞相比，Ad/IL24 感染的 U87 细胞中 caspase-3 和 TNF-α水平显著升高，而 Survivin 表达水平降低。Ad/IL-24 感染后肿瘤细胞中发现 TRAIL 上调，凋亡级联调节因子的研究表明，Ad/IL-24 可通过 TNF 家族死亡受体进一步增强凋亡的激活。在本研究中，我们证明 IL-24 表达可显著激活 P38 MAPK。此外，在 GBM 细胞中过表达 mda-7/IL-24 诱导自噬，这是通过 LC3-II 的上调触发的。
本研究表明 IL-24 对神经胶质瘤具有抗肿瘤作用，可能是 GBM 癌症基因治疗的一种有前途的治疗方法。
