obligate anaerobic salmonella typhimurium yb1could induce apoptosis of human neuroblastoma cells in vitro vivo without significant toxicity‹#›單擊此處編輯母

1、Neuroblastoma is an embryonal solid tumor of the autonomic nervous system. It arises in tissues of the sympathetic nervous system, typically in the adrenal medulla or paraspinal ganglia. Although many lower-stage neuroblastomas are encapsulated and can be surgically excised with little chance of com

2、plications, higher-stage tumors often infiltrate local organ structures, surround critical nerves and vessels such as the celiac axis, and are largely unresectable at the time of diagnosis. Neuroblastoma is a childhood cancer that is diagnosed at a median age of about 17 months. Tumors can arise any

3、where along the sympathetic nervous system, with the majority occurring in the adrenal medulla and paraspinal ganglia. Primary tumors in the neck or upper chest can cause Horners syndrome. Tumors along the spinal column can expand through the intraforaminal spaces and cause cord compression, with re

4、sulting paralysis. Neuroblastomas typically metastasize to regional lymph nodes and to the bone marrow. The tumor also can metastasize to the liver and lung. -John M. Maris, M.D. N Engl J Med 2010;362:2202-11.Hypoxia provokes a general adaptive response in neuroblastoma cells and confirm loss of the

5、 neuronal phenotype and gain of stem-cell characteristics such as self-renewal, tumor propagation and chemotherapeutic drug resistance. This cell subpopulation resides in the core of the solid tumor, where is hypoxic or anoxic. The cells were termed cancer stem cells(CSCs) by most groups. cancer ste

6、m cells reside within a perivascular niche. Regulated by oncogene activation or microenvironment conditions such as hypoxia, CSCs are strongly capable of stimulating the growth of the neovasculature by expressing high levels of pro-angiogenic factors such as VEGF. On the other hand, blood vessels cr

7、eate a vascular niche to help maintain CSC population. Such reciprocal relationships between CSCs and blood vessels enable rapid and sustained tumorigenesis. -Li Z, Wang H, Eyler CE,et cr. J Biol Chem. 2009 Jun 19;284(25):16705-9. Within the perivascular, newly formed blood vessels in tumors are hig

8、hly irregular and poorly organized, leading to the formation of multiple regions of hypoxia (1% oxygen) or anoxia (0% oxygen). The theraputic gene vectors and cytotoxic drugs can not penetrate the poorly vascularized, hypoxic regions of tumors, leaving these sites untreated. Moreover, hypoxic tumor

9、cells and CSCs are often non-proliferating and are therefore relatively refractory to many forms of chemotherapy. This prompted the search for a cellular vector, which can migrate into and thrive in hypoxic regions of solid tumors and is programmed to lyse in normal tissues, after systemic administr

10、ation.As a facultative anaerobe, Salmonella expresses a transcription factor, the Fumarate and Nitrate Reduction regulator (FNR), which responds to hypoxia and activates gene expression under anaerobic conditions. The asd gene encodes aspartate -semialdehyde dehydrogenase, an enzyme for synthesis of

11、 diaminopimelic acid (DAP), an essential component of the peptidoglycan of the cell wall of Gram- bacteria. In the absence of DAP, asd mutants undergo lysis. Since DAP is not present in mammalian tissues, this balanced-lethal system imposes a requirement for all living Salmonella to carry the recomb

12、inant asd+ plasmid. Construction of the hypoxia conditioned essential gene ASD expression cassette. The sense hypoxia promoter 1 contains the FNR regulated promoter and the aerobic promoter is the antisense promoter. This makes the engineered YB1 strain only survive in tumor core and is programmed t

13、o lyse in normal tissues.resultsFigure 1. The in vitro flow cytometry results of the annexin V assay after the co-culture with the 2 strains of Salmonella under the anaerobic and aerobic conditions. YB1 induced 3 folds higher apoptosis in anaerobic than in aerobic condition (31.4% vs 10.9%). Such di

14、fferential effect was not noted in SL7207 treated cells. In addition, the differential killing effect between YB1 and SL7202 under anaerobic condition was not significantly different in the in vitro short term culture setting. Figure 2. TLR4 & TLR5 expression of neuroblastoma cells increased under a

15、naerobic condition. The expression of TLR-4,5,9 genes on SK-NLP/luciferase cells after treated with YB1 and SL7207 under anaerobic and YB1 under aerobic conditions. The data were pooled from the experiments done in triplicate, and expressed as mean SD. * P 0.05; * P 0.01.Figure 3. The ICH results fr

16、om the TLR4 and 5 expression in the tumor. TLR4 stain: A and B; TLR 5 stain: C and D; YB1 treated tumor: B and D; control: A and C.Figure 4. Preferential accumulation of YB1 in the orthotopic tumors from mice administered systemically. SCID/beige mice bearing orthotopic SK-NLP/luciferase tumors were

17、 injected through tail vein with YB1 (5107 CFU) at day 14. The CFU amounts of accumulated YB1 (means SD, n =6) in the tumors, hearts, livers, and spleens were determined at day 28. *P 0.01.Figure 5. The immunohistochemical staining analysis of YB1 distribution. After 2 weeks treatment with YB1, SCID

18、/beige mice were euthanized and YB1 accumulation in tumors (A and B), hearts (C and D), livers (E and F) and spleens (G and H) were assessed in tissue sections by immuno-staining (brown color pointed with red arrow). The left panel was YB1 t r e a t e d g r o u p , t h e r i g h t p a n e l w a s c

19、o n t r o l g r o u p .Figure 6. The percentage of body weight loss of the mice after the injection of YB1 and PBS. The green line panels belonged to the YB1 treated group, and the pink line panels belonged to the control group. The body weight was observed every two days. In general, the body weigh

20、t loss among the YB1 treated group was more than that of the control group during the initial 8 days after the YB1 injection (each p0.05), especially during the initial 6 days (each p0.01). The p values of each time point were showed above. a bFigure 7. Monitoring of in situ neuroblastoma growth by

21、Xenogen IVIS 100 at different time points after SK-NLP/luciferase cells implantation and YB1 treatment. The figure 7a was taken after 2 weeks of neuroblastoma implantation and before the bacterial inoculation. The upper panel was assigned to YB1 treatment and the lower panel represented the control

22、group. Figure 7b was taken 2 weeks after YB1 and PBS injection. The intensity of luciferin signals significantly decreased, not only at the primary tumor but also at the metastatic sites. For treated mouse 5, there was no more luciferin signal detected.Figure 8. Decrease in tumors size of neuroblastoma after YB1 treatment. The upper panel was YB1 treated group, the