Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb 4;7(2):423-38.
doi: 10.3390/toxins7020423.

The anti-cancer potency and mechanism of a novel tumor-activated fused toxin, DLM

Dejun Sun  1 , Miaonan Sun  2 , Wenhe Zhu  3 , Zhiding Wang  4 , Yuefei Li  5 , Jie Ma  6
Affiliations

The anti-cancer potency and mechanism of a novel tumor-activated fused toxin, DLM

Dejun Sun et al. Toxins (Basel). .

Abstract

Melittin, which acts as a membrane-disrupting lytic peptide, is not only cytotoxic to tumors, but also vital to normal cells. Melittin had low toxicity when coupled with target peptides. Despite significant research development with the fused toxin, a new fused toxin is needed which has a cleavable linker such that the fused toxin can release melittin after protease cleavage on the tumor cell surface. We describe a novel fused toxin, composed of disintegrin, uPA (urokinase-type plasminogen activator)-cleavable linker, and melittin. Disintegrin is a single strand peptide (73 aa) isolated from Gloydius Ussuriensis venom. The RGD (Arg-Gly-Asp) site of disintegrin dominates its interaction with integrins on the surface of the tumor cells. uPA is over-expressed and plays an important role in tumor cell invasiveness and metastatic progression. The DLM (disintegrin-linker-melittin) linker is uPA-cleavable, enabling DLM to release melittin. We compared binding activity of our synthesized disintegrin with native disintegrin and report that DLM had less binding activity than the native form. uPA-cleavage was evaluated in vitro and the uPA-cleavable linker released melittin. Treating tumors expressing uPA with DLM enhanced tumor cell killing as well as reduced toxicity to erythrocytes and other non-cancerous normal cells. The mechanism behind DLM tumor cell killing was tested using a DNA ladder assay, fluorescent microscopy, flow cytometry, and transmission electron microscopy. Data revealed tumor cell necrosis as the mechanism of cell death, and the fused DLM toxin with an uPA-cleavable linker enhanced tumor selectivity and killing ability.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Degradation of DLM by uPA. Lane 1: Protein standard; Lane 2: DLM (12,000 Da, Table S1); Lane 3: DLM cleavage by uPA (band ~10,000 Da, and 73 + 16 aa), indicating that DLM can be cleaved by uPA.
Figure 2
Figure 2
DLM and erythrocytes. Free melittin and DLM activated by uPA are hemolytic, but DLM is weakly hemolytic. Results are expressed as means ± standard error.
Figure 3
Figure 3
Retention of biological activity of disintegrin in DLM. Results are expressed as mean ± standard error.
Figure 4
Figure 4
DLM binding to washed platelets compared with disintegrin. Results are expressed as means ± standard error.
Figure 5
Figure 5
MCF-7 cell survival after treatment with serial dilutions of DLM.
Figure 6
Figure 6
MCF-7 cells after gel electrophoresis fluorescence of DNA after 48 h. Lane 1: Negative control; Lane 2: MCF-7 cells treated with 0.164 μM DLM; Lane 3: 0.328 μM DLM; Lane 4: 0.626 μM DLM; Lane 5: 1.313 μM DLM; Lane 6: 2.626 μM DLM; Lane 7: 10 μM cis-platinum; Lane 8: 2.626 μM melittin; Lane 9: DNA marker.
Figure 7
Figure 7
Cytotoxicity of DLM to MCF-7 measured by flow cytometry. (A) controls; (B) 0.164 μM DLM; (C) 0.328 μM DLM; (D) 0.656 μM DLM; (E) 1.313 μM DLM.
Figure 7
Figure 7
Cytotoxicity of DLM to MCF-7 measured by flow cytometry. (A) controls; (B) 0.164 μM DLM; (C) 0.328 μM DLM; (D) 0.656 μM DLM; (E) 1.313 μM DLM.
Figure 8
Figure 8
Cytotoxicity to MCF-7 observed under fluorescent microscopy (200×). (A,a) controls; (B,b) 0.328 μM DLM; (C,c) 0.656 μM DLM; (D,d) 1.313 μM DLM.
Figure 8
Figure 8
Cytotoxicity to MCF-7 observed under fluorescent microscopy (200×). (A,a) controls; (B,b) 0.328 μM DLM; (C,c) 0.656 μM DLM; (D,d) 1.313 μM DLM.
Figure 9
Figure 9
MCF-7 cells under TEM. (a) Control MCF-7 cell under TEM. A: Cells are larger with microvillus and the nucleus is large and irregular (2000×); B: Some cells have free ribosomes (12,000×); (b) MCF-7 cells treated with 0.328 μM DLM under TEM. A: Cells are smaller and leaky, the cytoplasm has many vacuoles and few lipid droplets (5000×); B: The ruptured membrane (indicated by arrow), released contents and degraded organelles are present in the cytoplasm. ER, nuclear matrix, and cytoplasm have vacuoles (7500×); (c) MCF-7 cell treated with 0.656 μM DLM under TEM. A: Cytoplasm shows membrane rupture (indicated by arrow at top) and the nucleus has vacuoles (indicated by arrow at right). Intact organelles are scarce. (6000×); B: Cells are swollen and the nuclear matrix (indicated by arrow) and the cytoplasm have vacuoles (6000×).
Figure 9
Figure 9
MCF-7 cells under TEM. (a) Control MCF-7 cell under TEM. A: Cells are larger with microvillus and the nucleus is large and irregular (2000×); B: Some cells have free ribosomes (12,000×); (b) MCF-7 cells treated with 0.328 μM DLM under TEM. A: Cells are smaller and leaky, the cytoplasm has many vacuoles and few lipid droplets (5000×); B: The ruptured membrane (indicated by arrow), released contents and degraded organelles are present in the cytoplasm. ER, nuclear matrix, and cytoplasm have vacuoles (7500×); (c) MCF-7 cell treated with 0.656 μM DLM under TEM. A: Cytoplasm shows membrane rupture (indicated by arrow at top) and the nucleus has vacuoles (indicated by arrow at right). Intact organelles are scarce. (6000×); B: Cells are swollen and the nuclear matrix (indicated by arrow) and the cytoplasm have vacuoles (6000×).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Skeel R.T. Handbook of Cancer Chemotherapy. 6th ed. Lippincott Williams & Wilkins; Philadelphia, PA, USA: 2003.
    1. Takimoto C.H., Calvo E. Principles of oncologic pharmacotherapy. In: Pazdur R., Wagman L.D., Camphausen K.A., Hoskins W.J., editors. Cancer Management: A Multidisciplinary Approach. 11th ed. CMP United Business Media; Manhasset, NY, USA: 2009.
    1. Moertel C.G., Fleming T.R., Macdonald J.S., Haller D.G., Laurie J.A., Goodman P.J., Ungerleider J.S., Emerson W.A., Tormey D.C., Glick J.H., et al. Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma. N. Engl. J. Med. 1990;322:352–358. doi: 10.1056/NEJM199002083220602. - DOI - PubMed
    1. Sikora K., Advani S., Koroltchouk V., Magrath I., Levy L., Pinedo H., Schwartsmann G., Tattersall M., Yan S. Essential drugs for cancer therapy: A world health organization consultation. Ann. Oncol. 1999;10:385–390. doi: 10.1023/A:1008367822016. - DOI - PubMed
    1. Soler M., González-Bártulos M., Soriano-Castell D., Ribas X., Costas M., Tebar F., Massaguer A., Feliu L., Planas M. Identification of BP16 as a non-toxic cell-penetrating peptide with highly efficient drug delivery properties. Org. Biomol. Chem. 2014;12:1652–1663. doi: 10.1039/c3ob42422g. - DOI - PubMed

Publication types

MeSH terms