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Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis.

Jiang WG, Lu XA, Shang BY, Fu Y, Zhang SH, Zhou D, Li L, Li Y, Luo Y, Zhen YS - BMC Cancer (2013)

Bottom Line: ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability.Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.The ES-based fusion protein therapy provides some fundamental information for further drug development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P, R, China. yluo@tsinghua.edu.cn.

ABSTRACT

Background: Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety.

Methods: In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer.

Results: ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.

Conclusions: The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin-based fusion proteins and their enediyne-energized analogs probably provides a promising modality in cancer therapy.

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HMEC and 4T1 migration in wound healing assay using ES or ES-based fusion proteins as inhibitors. Pictures were taken at magnification 40X in light microscopy (A and C). Quantification results of migrated cells, counted using the software Image-Pro Plus 6.0, are shown in B and D, assuming control as 100%. Inhibitors were used at indicated concentration. Results shown are average values of 6 representative fields in each of the two different experiments performed in duplicates, and error bars represent SEM. *, P ≤ 0.05, **, P ≤ 0.001, compared with ES, respectively. Cells were viewed with a microscope and pictures were taken at × 40.
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Figure 2: HMEC and 4T1 migration in wound healing assay using ES or ES-based fusion proteins as inhibitors. Pictures were taken at magnification 40X in light microscopy (A and C). Quantification results of migrated cells, counted using the software Image-Pro Plus 6.0, are shown in B and D, assuming control as 100%. Inhibitors were used at indicated concentration. Results shown are average values of 6 representative fields in each of the two different experiments performed in duplicates, and error bars represent SEM. *, P ≤ 0.05, **, P ≤ 0.001, compared with ES, respectively. Cells were viewed with a microscope and pictures were taken at × 40.

Mentions: New blood vessel formation requires that the endothelial cells migrate towards the sources of growth factor. We used the HMEC wound healing assay to observe the ability of ES-based fusion proteins in inhibiting endothelial cell migration. As shown in Figure 2A, cells were able to migrate towards the wound area in higher number when exogenous rhVEGF was added. ES or ES-based fusion proteins all demonstrated the ability of inhibiting HMEC migration at different concentrations when compared with rhVEGF control (Figure 2A). Comparison of quantified results shows that ES-based fusion proteins are more potent than ES, and ES-LDP exhibits a stronger inhibitory effect than LDP-ES (Figure 2B). These results indicate that ES-based fusion proteins have increased capability in inhibiting VEGF-induced endothelial cell migration.


Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis.

Jiang WG, Lu XA, Shang BY, Fu Y, Zhang SH, Zhou D, Li L, Li Y, Luo Y, Zhen YS - BMC Cancer (2013)

HMEC and 4T1 migration in wound healing assay using ES or ES-based fusion proteins as inhibitors. Pictures were taken at magnification 40X in light microscopy (A and C). Quantification results of migrated cells, counted using the software Image-Pro Plus 6.0, are shown in B and D, assuming control as 100%. Inhibitors were used at indicated concentration. Results shown are average values of 6 representative fields in each of the two different experiments performed in duplicates, and error bars represent SEM. *, P ≤ 0.05, **, P ≤ 0.001, compared with ES, respectively. Cells were viewed with a microscope and pictures were taken at × 40.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016579&req=5

Figure 2: HMEC and 4T1 migration in wound healing assay using ES or ES-based fusion proteins as inhibitors. Pictures were taken at magnification 40X in light microscopy (A and C). Quantification results of migrated cells, counted using the software Image-Pro Plus 6.0, are shown in B and D, assuming control as 100%. Inhibitors were used at indicated concentration. Results shown are average values of 6 representative fields in each of the two different experiments performed in duplicates, and error bars represent SEM. *, P ≤ 0.05, **, P ≤ 0.001, compared with ES, respectively. Cells were viewed with a microscope and pictures were taken at × 40.
Mentions: New blood vessel formation requires that the endothelial cells migrate towards the sources of growth factor. We used the HMEC wound healing assay to observe the ability of ES-based fusion proteins in inhibiting endothelial cell migration. As shown in Figure 2A, cells were able to migrate towards the wound area in higher number when exogenous rhVEGF was added. ES or ES-based fusion proteins all demonstrated the ability of inhibiting HMEC migration at different concentrations when compared with rhVEGF control (Figure 2A). Comparison of quantified results shows that ES-based fusion proteins are more potent than ES, and ES-LDP exhibits a stronger inhibitory effect than LDP-ES (Figure 2B). These results indicate that ES-based fusion proteins have increased capability in inhibiting VEGF-induced endothelial cell migration.

Bottom Line: ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability.Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.The ES-based fusion protein therapy provides some fundamental information for further drug development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P, R, China. yluo@tsinghua.edu.cn.

ABSTRACT

Background: Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety.

Methods: In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer.

Results: ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.

Conclusions: The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin-based fusion proteins and their enediyne-energized analogs probably provides a promising modality in cancer therapy.

Show MeSH
Related in: MedlinePlus