Inhibitory effect of Bifidobacterium infantis-mediated sKDR prokaryotic expression system on angiogenesis and growth of Lewis lung cancer in mice

Abstract

BackgroundTo construct the Bifidobacterium infantis-mediated soluble kinase insert domain receptor (sKDR) prokaryotic expression system and to observe its inhibitory effect on growth of human umbilicus vessel endothelial cells (HUVECs) in vitro and Lewis lung cancer (LLC) on mice in vivo.MethodsThe Bifidobacterium infantis-mediated sKDR prokaryotic expression system was constructed through electroporation and subsequently identified through PCR and Western blot analysis. HUVECs were added to the products of this system to evaluate the anti-angiogenesis effect through MTT assay in vitro. The LLC mice models were divided into three groups: one group treated with saline (group a); one group treated with recombinant Bifidobacterium infantis containing pTRKH2-PsT plasmid group (group b); and one group treated with recombinant Bifidobacterium infantis containing pTRKH2-PsT/sKDR plasmid group (group c). The quality of life and survival of mice were recorded. Tumor volume, tumor weight, inhibitive rate, and necrosis rate of tumor were also evaluated. Necrosis of tumor and signals of blood flow in tumors were detected through color Doppler ultrasound. In addition, microvessel density (MVD) of the tumor tissues was assessed through CD31 immunohistochemical analysis.ResultsThe positively transformed Bifidobacterium infantis with recombinant pTRKH2-PsT/sKDR plasmid was established, and was able to express sKDR at gene and protein levels. The proliferation of HUVECs cultivated with the extract of positively transformed bacteria was inhibited significantly compared with other groups (P < 0. 05). The quality of life of mice in group c was better than in group a and b. The recombinant Bifidobacterium infantis containing pTRKH2-PsT/sKDR plasmid enhanced the efficacy of tumor growth suppression and prolongation of survival, increased the necrosis rate of tumor significantly, and could obviously decrease MVD and the signals of blood flow in tumors.ConclusionThe Bifidobacterium infantis-mediated sKDR prokaryotic expression system was constructed successfully. This system could express sKDR at gene and protein levels and significantly inhibit the growth of HUVECs induced by VEGF in vitro. Moreover, it could inhibit tumor growth and safely prolong the survival time of LLC C57BL/6 mice.

Estrogen and insulin‐like growth factor 1 synergistically promote the development of lung adenocarcinoma in mice

“…For cDNA synthesis, RNA of equal volume was collected from each group for reverse transcription. The components of the mixture for RT‐PCR are presented as described previously: RNA, 5 μl; ligo(dT) 20 (10 mM) 1 μl; dNTP (10 mM) 2 μl; ddH 2 O (RNase free) up to 19; conditions, 70°C, 5 min, transiently on ice; and above mixture, 19 μl; 5× RT buffer, 5 μl; HRP(RRI)/RNase inhibitor, 0.5 μl; M‐MLV, 0.5 μl; ddH 2 O (RNase free), up to 25; conditions, 42°C, 60 min, 95°C for 5 min.…”

The Cross-Talk Between Gut Microbiota and Lungs in Common Lung Diseases

“…Bifidobacterium infantis-mediated sFlt-1 gene transferring system showed suppression on the tumor growth and longer survival time in C57BL/6 mice with lung cancer (Zhu et al, 2011). Bifidobacterium infantismediated soluble kinase insert domain receptor inhibited the tumor growth via increasing the necrosis rate of the tumor (Li et al, 2012). However, negative effect of microbial agents on cancer development has also been pointed out due to the potentially oncogenic toxins and metabolites by bacteria.…”

Bifidobacteria Expressing Tumstatin Protein for Antitumor Therapy in Tumor-Bearing Mice

“…AF258351; Figure 1A). Plasmids pBBAD-Tum and pBBAD-GFP were transformed into B longum NCC 2705 using electroporation with a Gene Pulser and Pulse Controller apparatus (Bio-Rad, Hercules, California) at 2.5 kV, 25 Mf, and 200 O. 22 The transformed bacteria were plated on 24 MRS agar supplemented with 0.05% L-cysteine and 100 mg/ mL ampicillin, and incubated anaerobically at 37 C for 48 hours.…”

“…The Gramstained section was evaluated for BL-GFP or BL-Tum. The MVD of tumor tissue was assessed according to the method of Weidner et al 25 The immuno-stained sections were initially screened at low magnification (40Â) to identify hot spots, which were the areas with the highest levels of neovascularization. Endothelial cells or endothelial cell clusters that were stained yellow-brown and clearly separated from adjacent microvessels, tumor cells, and other connective tissues were considered countable microvessels.…”