Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

Horibe, Tomohisa; Torisawa, Aya; Akiyoshi, Ryutaro; Hatta‐Ohashi, Yoko; Suzuki, Hirobumi; Kawakami, Koji

doi:10.1002/bio.2508

Cited by 35 publications

(44 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Kojic acid and rutin were purchased from WAKO (Osaka, Japan). The human glioblastoma U251 cell line was obtained as reported previously . U251 cells stably transfected with pBipPro‐Luc (U251/Luc), in which the promoter region of Bip was cloned into the pGL4.14 vector (Promega, Madison, WI, USA), were established previously .…”

Section: Methodsmentioning

confidence: 99%

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real‐time bioluminescence imaging

et al. 2017

Self Cite

View full text Add to dashboard Cite

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods.

show abstract

Section: Methodsmentioning

confidence: 99%

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real‐time bioluminescence imaging

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…Since our previous study showed that bioluminescence imaging at the single-cell level with the LV200 imaging system is a powerful tool for analyzing gene expression (based on a reporter assay specific to cells with low transfection efficiency) [23], we applied this imaging technique to evaluate the effect of each treatment on Hsp70 expression. Although the transfection efficiency of reporter plasmid DNA in BT20 was not so high compared with that of other cancer cells with high transfection efficiency, such as HeLa cells [23], analyzing Hsp70 promoter activation on single-cell imaging proved to be effective for time-course monitoring of this promoter in living cancer cells.…”

Section: Discussionmentioning

confidence: 99%

“…Although the transfection efficiency of reporter plasmid DNA in BT20 was not so high compared with that of other cancer cells with high transfection efficiency, such as HeLa cells [23], analyzing Hsp70 promoter activation on single-cell imaging proved to be effective for time-course monitoring of this promoter in living cancer cells. Treatment with Antp-TPR with or without R11-Hsp70 did not activate the Hsp70 promoter in breast cancer cells, whereas treatment with 17-AAG did (Figure 4C).…”

Section: Discussionmentioning

confidence: 99%

“…Luminescence images at the single-cell level were obtained using the LV200 luminescence imaging system (Olympus) as described previously [22, 23]. Briefly, the dish was kept at 37°C in a humidified chamber and images were taken with a 40× objective at 5-min intervals with an exposure of 10 s while observing promoter activity after the addition of D-luciferin (Promega) at a final concentration of 500 μM.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide

et al. 2014

Self Cite

View full text Add to dashboard Cite

BackgroundHeat shock protein (Hsp) 90 and Hsp70 are indispensable for cell survival under conditions of stress. They bind to client proteins to assist in protein stabilization, translocation of polypeptides across the cell membrane, and recovery of proteins from aggregates in the cell. Therefore, these proteins have recently emerged as important targets in the treatment of cancer. We previously reported that the newly designed Antp-TPR hybrid peptide targeting Hsp90 induced cytotoxic activity to cancer cells both in vitro and in vivo.MethodsTo further improve the cytotoxic activity of Antp-TPR toward cancer cells, we investigated the effect of a Hsp70-targeted peptide, which was made cell-permeable by adding the polyarginine with a linker sequence, on the cytotoxic activity of Antp-TPR in breast cancer cell lines.ResultsIt was revealed that Antp-TPR in the presence of a Hsp70-targeted peptide induced effective cytotoxic activity toward breast cancer cells through the descrease of Hsp90 client proteins such as p53, Akt, and cRaf. Moreover, the combined treatment with these peptides did not induce the up-regulation of Hsp70 protein, as determined by western blotting, a promoter assay using a luminometer, and single-cell level imaging with the LV200 system, although a small-molecule inhibitor of Hsp90, 17-allylamino-demethoxygeldanamycin (17-AAG), did induce the up-regulation of this protein. We also found that treatment with Antp-TPR, Hsp70-targeted peptide, or a combination of the two did not induce an increase in the glutathione concentrations in the cancer cells.ConclusionThese findings suggest that targeting both Hsp90 and Hsp70 with Antp-TPR and Hsp70-targeted peptide is an attractive approach for selective cancer cell killing that might provide potent and selective therapeutic options for the treatment of cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-615) contains supplementary material, which is available to authorized users.

show abstract

“…ATP dynamics in cancer cells were measured after treatment with the hybrid peptide by single‐cell bioluminescence imaging using the LV200 system (Olympus) as described previously (Akiyoshi & Suzuki, ; Horibe et al., ; Kikuchi et al., ). Briefly, U251 cells stably transfected with firefly luciferase‐containing reporter plasmids of the cytomegalovirus (CMV) promoter pGL4.50 (U251/CMVLuc; Promega) were prepared after the transient transfection with Lipofectamine LTX in selective medium containing 200 μg/ml hygromycin B (Nacalai Tesque), and bioluminescence images were obtained with the LV200 system.…”

Section: Methodsmentioning

confidence: 99%

A novel interleukin‐13 receptor alpha 2‐targeted hybrid peptide for effective glioblastoma therapy

et al. 2019

Self Cite

View full text Add to dashboard Cite

We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic‐type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin‐13 receptor alpha 2 (IL‐13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL‐13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL‐13Rα2–lytic hybrid peptide. The IL‐13Rα2–lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL‐13Rα2–lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin‐13 and induced rapid ATP dynamics in GB cells. Anti‐tumor activity of the IL‐13Rα2–lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL‐13Rα2–lytic hybrid peptide might be a potent therapeutic option for patients with GB.

show abstract

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

Cited by 35 publications

References 20 publications

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real‐time bioluminescence imaging

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real‐time bioluminescence imaging

Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide

A novel interleukin‐13 receptor alpha 2‐targeted hybrid peptide for effective glioblastoma therapy

Contact Info

Product

Resources

About