Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency

• Main Authors: Howell Stephen B, Manorek Gerald, Lin Xinjian, Yuan Xiaoqin
• Format: Article
• Language: English
• Published: BMC 2011-02-01
• Series: BMC Cancer
• Online Access: http://www.biomedcentral.com/1471-2407/11/61

<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 G₄S linker. This CPE-G₄S-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 (R₉) was introduced between the CPE and the rGel. CPE-R₉-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E₉) through a G₄S linker to R₉-rGel (E₉-G₄S-R₉-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R₉motif. However, introduction of CPE to the E₉-G₄S-R₉-rGel fusion protein (CPE-E₉-G₄S-R₉-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E₉-G₄S-R₉-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R₉-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E₉-G₄S-R₉-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 R₉non-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 R₉-rGel from CPE-E₉-G₄S-R₉-rGel.</p>