Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency
<p>Abstract</p> <p>Background</p> <p>Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challeng...
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doaj-f2575a190429422f83749b6400cebd4e2020-11-24T22:16:23ZengBMCBMC Cancer1471-24072011-02-011116110.1186/1471-2407-11-61Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potencyHowell Stephen BManorek GeraldLin XinjianYuan Xiaoqin<p>Abstract</p> <p>Background</p> <p>Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of <it>C. perfringens </it>enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008.</p> <p>Results</p> <p>CPE was fused to rGel at its N-terminal end via a flexible G<sub>4</sub>S linker. This CPE-G<sub>4</sub>S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R<sub>9</sub>) was introduced between the CPE and the rGel. CPE-R<sub>9</sub>-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E<sub>9</sub>) through a G<sub>4</sub>S linker to R<sub>9</sub>-rGel (E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R<sub>9 </sub>motif. However, introduction of CPE to the E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel fusion protein (CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R<sub>9</sub>-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel.</p> <p>Conclusion</p> <p>We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R<sub>9 </sub>non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R<sub>9</sub>-rGel from CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel.</p> http://www.biomedcentral.com/1471-2407/11/61 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Howell Stephen B Manorek Gerald Lin Xinjian Yuan Xiaoqin |
spellingShingle |
Howell Stephen B Manorek Gerald Lin Xinjian Yuan Xiaoqin Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency BMC Cancer |
author_facet |
Howell Stephen B Manorek Gerald Lin Xinjian Yuan Xiaoqin |
author_sort |
Howell Stephen B |
title |
Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency |
title_short |
Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency |
title_full |
Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency |
title_fullStr |
Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency |
title_full_unstemmed |
Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency |
title_sort |
challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency |
publisher |
BMC |
series |
BMC Cancer |
issn |
1471-2407 |
publishDate |
2011-02-01 |
description |
<p>Abstract</p> <p>Background</p> <p>Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of <it>C. perfringens </it>enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008.</p> <p>Results</p> <p>CPE was fused to rGel at its N-terminal end via a flexible G<sub>4</sub>S linker. This CPE-G<sub>4</sub>S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R<sub>9</sub>) was introduced between the CPE and the rGel. CPE-R<sub>9</sub>-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E<sub>9</sub>) through a G<sub>4</sub>S linker to R<sub>9</sub>-rGel (E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R<sub>9 </sub>motif. However, introduction of CPE to the E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel fusion protein (CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R<sub>9</sub>-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel.</p> <p>Conclusion</p> <p>We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R<sub>9 </sub>non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R<sub>9</sub>-rGel from CPE-E<sub>9</sub>-G<sub>4</sub>S-R<sub>9</sub>-rGel.</p> |
url |
http://www.biomedcentral.com/1471-2407/11/61 |
work_keys_str_mv |
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