Department of Otorhinolaryngology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, People's Republic of China.
Clinical Research Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, People's Republic of China.
PLoS One. 2020 Aug 3;15(8):e0237098. doi: 10.1371/journal.pone.0237098. eCollection 2020.
The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 μg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 μg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy.
表皮生长因子受体靶向癌症治疗通常面临耐药性问题，主要是由于基因突变。针对表皮生长因子保守序列的基因治疗用人工 microRNA 可能会避免此类问题。在这项研究中，我们构建了一种表达表皮生长因子靶向人工 microRNA 和活性 revCASP3 的重组腺病毒（Ad-EC），受肿瘤特异性 SLPI 启动子的控制，并评估了其在体外和体内对 HEP-2 癌细胞的抑制作用。MTT 检测显示，在 MOI50 时，Ad-EC 组的细胞生长抑制率在 72 小时时为 44.0%，而对照病毒 Ad-GFP 组为 7.7%，西妥昔单抗（500μg/ml）组为 3.6%。流式细胞术分析显示，Ad-EC 组晚期凋亡细胞率为 36.1%，明显高于 Ad-GFP 组的 6.5%（p<0.001）。当 Ad-EC（MOI50）与 CDDP（0.25μg/ml）联合使用时，晚期凋亡细胞率增加到 61.2%，明显高于单独用药组（P<0.001）。实时 xCELLigence 系统记录到 Ad-EC 和 CDDP 组的有效细胞生长抑制作用，并且 Ad-EC 加 CDDP 组的抑制作用增强。Western blot 显示，Ad-EC 可抑制 AKT 通路和 ERK1/2 通路的激活，而西妥昔单抗使 AKT 通路过度激活。在裸鼠 HEP-2 异种移植的体内实验中，与 PBS 组相比，Ad-EC、CDDP 和 Ad-EC 加 CDDP 组的肿瘤抑制作用更为明显（P<0.01）。综上所述，这些数据支持这种新型基因治疗策略对癌细胞的有效抑制作用。
