Abstract
BackgroundTreatment 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 C. perfringens 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.ResultsCPE 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 R9 motif. 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.ConclusionWe 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 R9 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 R9-rGel from CPE-E9-G4S-R9-rGel.
Highlights
Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment
Vectors expressing other variants containing combinations of C. perfringens enterotoxin (CPE), E9, G4S and R9 were constructed by splicing overlap extension PCR (SOE-PCR)
Following expression in E. coli, purification and SUMO cleavage to remove the 6×His tag, the final purified products of all the recombinant gelonin (rGel)-based fusion toxins migrated on SDS-PAGE at the expected molecular weights of 28.2, 31.9, 33.0, 29.6, 31.3, and 34.4 kDa, respectively, for rGel, CPE-G4S-rGel, CPE-R9-rGel, R9-rGel, E9-G4S-R9-rGel and CPE-E9-G4SR9-rGel (Figure 1B)
Summary
Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. Some examples include increased expression of CLDN3 and CLDN4 in prostate and uterine cancers [4,5], and high CLDN4 expression in pancreatic cancer [6,7] These two genes are not normally highly expressed in non-malignant human tissues including the normal ovarian epithelium [8], clearly associating abundance of these two proteins with malignancy. Their functional role in cancer development and progression remains unclear, the differential expression of these proteins between tumor and normal cells makes them prime candidates for cancer targeted therapy [9]
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