Wang Changdong, Ma Yongping, Hu Qiongwen, Xie Tingting, Wu Jiayan, Zeng Fan, Song Fangzhou
Department of Biochemistry & Molecular Biology, Molecular Medicine & Cancer Research Center, Chongqing Medical University, Yuzhong District, Yi XueYuan Road, No 1, Chongqing, 400016, People's Republic of China.
BMC Cancer. 2016 Jul 27;16:545. doi: 10.1186/s12885-016-2608-3.
Directly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms.
Bifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression.
The results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy.
BF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim without inducing necroptosis and autophagy. Furthermore, BF-rTK + GCV showed to repress the inflammation of tumor through downregulating TNF-α expression. Survival analysis results of multiple cancer models confirmed that BF-rTK + GCV system has a wide field of application in solid tumor gene therapy.
将治疗性自杀基因直接靶向实体瘤是癌症基因治疗的一种有前景的方法。利用有效的自杀基因载体治疗实体瘤仍然是一项挑战。鉴于缺乏有效的治疗方法，我们构建了双歧杆菌重组胸苷激酶（BF-rTK）-更昔洛韦（GCV）靶向系统（BKV）以满足这一需求并探索抗肿瘤机制。
在人结肠320肠道异种移植瘤模型中，瘤内注射双歧杆菌（BF）或BF-rTK，同时给予或不给予更昔洛韦。使用凋亡抗体阵列、实时PCR和蛋白质印迹法分析肿瘤组织。通过基因沉默方法分析结肠320细胞。还在结肠320细胞中检测自噬和坏死性凋亡。同时，采用三种人类消化系统异种移植瘤模型（结直肠癌结肠320、胃癌MKN-45和肝癌SSMC-7721）和一种乳腺癌（MDA-MB-231）模型来验证BF-rTK + GCV在实体瘤基因治疗中的普遍性。在瘤内给予BF-rTK + GCV后，评估三种人类癌症模型的存活率。对肿瘤中炎症标志物（TNF-α）的分析表明，BF-rTK + GCV显著抑制TNF-α表达。
结果表明，BF-rTK + GCV诱导肿瘤凋亡，未发生自噬和坏死性凋亡。凋亡由FasL和TNFR2介导的多种信号通路转导，主要通过Bid和Bim激活线粒体凋亡调控，通过沉默Bid或/和Bim可挽救该过程。然而，BF + GCV仅通过Fas/FasL信号通路诱导凋亡，并伴有P53表达增加。我们进一步发现，BF-rTK + GCV抑制炎症标志物TNF-α的表达。然而，BF-rTK + GCV未导致坏死性凋亡和自噬。
BF-rTK + GCV通过Bid和Bim介导的线粒体凋亡调控，由FasL和TNFR2介导诱导肿瘤凋亡，未诱导坏死性凋亡和自噬。此外，BF-rTK + GCV通过下调TNF-α表达显示出抑制肿瘤炎症的作用。多种癌症模型的生存分析结果证实，BF-rTK + GCV系统在实体瘤基因治疗中具有广泛的应用前景。
