Authors: Xiaoqin Yuan (equal contributor) [1]; Xinjian Lin (equal contributor) [1]; Gerald Manorek [1]; Stephen B Howell (corresponding author) [1]
Background
The claudin (CLDN) family of transmembrane proteins plays an integral role in the formation and function of tight junctions. Using gene expression profiling, we and others have found that claudin-3 (CLDN3) and claudin-4 (CLDN4) genes are highly expressed in ovarian cancers [1, 2, 3]. In addition, several other studies have reported aberrant claudin expression in various cancers. 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. Although 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]. Preclinical studies have shown that tumor cells over-expressing CLDNs can be successfully targeted both in vitro and in vivo by a fusion protein composed of the C-terminal fragment (amino acids 184 to 319) of Clostridium perfringens enterotoxin (CPE), a natural ligand for CLDNs, and the protein synthesis inhibitory factor (PSIF) which lacks the cell binding domain of Pseudomonas exotoxin [10, 11]. When CPE binds to CLDNs it triggers endocytosis most likely via a clathrin-dependent process. We previously reported in vitro characterization of a fusion protein, CPE[sub.290-319]-TNF, and demonstrated that the C-terminal 30 amino acids (amino acids 290-319) of CPE could effectively target TNF to ovarian cancer cells expressing claudin-3 and claudin-4 [12].
Gelonin (rGel) is a class I ribosome-inactivating protein derived from the plant Gelonium multiforum. Similar in action to other plant toxins such as ricin, gelonin induces cell death by removing the base A4324 in 28 s rRNA which prevents the association of elongation factor-1 and -2 (EF-1 and EF-2) with the 60 s ribosomal subunit, eventually causing cell death in eukaryotic cells [13]. Since gelonin functions enzymatically, only a few molecules are needed to kill a cell, but by itself gelonin has very limited toxicity because it is not able to cross the plasma membrane at levels that are therapeutically useful. This has prompted the development of strategies to improve intracellular accumulation. Gelonin has been used to construct a large number of different kinds of immunotoxins, some of which are currently undergoing clinical testing [14, 15, 16].
Cancer therapies that exploit targeting ligands to deliver attached cytotoxic drugs selectively to malignant cells are currently receiving significant attention. However, the lipophilic nature of the biological membranes restricts the direct intracellular delivery of such compounds. While some short peptides can enter cells, the cell membrane prevents large molecules, such as proteins and DNA, from entering cells unless there is an active transport mechanism. Under certain circumstances these molecules, or even small particles, can be transferred from the extracellular space into cells by the receptor-mediated endocytosis. However, the problem is that most molecules or particles entering the cell via the endocytic pathway become entrapped in endosomes and eventually get degraded in the lysosomal compartment. As a result, only a small fraction of active material reaches the cytoplasm. It has been reported that a poly-arginine tract such as R[sub.9], which is also a furin cleavage site, can aid in translocating a recombinant pro-apoptotic protein targeting the HER2 receptor from the endosomal to the cytosolic compartment leading to enhanced cell killing activity [17]. However, due to the fact that all the arginine-rich cell-penetrating peptides (CPPs) induce a strong non-specific cell binding, they lack cell specificity and this remains the major impediment to development. Tsien and coworkers [18] previously developed a new strategy, designated "activatable cell penetrating peptides (ACPP)" by which the cellular association of the positively charged R[sub.9 ]is effectively blocked by fusing it to a domain made up of negatively charged glutamates (E[sub.9]) via a cleavable linker. Adsorption and cellular uptake of the CPP portion and its attached cargo are inhibited until the linker is cleaved by a tumor protease. In the present study, we describe the construction and characterization of several rGel-based chimeric toxins composed of different combinations of CPE[sub.290-319], R[sub.9 ]and E[sub.9]. We have used these to examine how these modules affect the internalization and cytotoxic activity when tested against CLDN-expressing ovarian cancerous cells.
Methods
Reagents
Tissue culture media were purchased from Life Technologies (Frederick, MD), pE-SUMOstar vector and SUMOstar protease 1 from LifeSensors, Inc (Malvern, PA), and metal-affinity resin Ni-NTA agarose from Qiagen (Valencia, CA). Rabbit anti-gelonin antibody was a gift from Dr. Michael G. Rosenblum (MD Anderson Cancer Center, Houston, TX). Texas red-labeled secondary antibodies against rabbit immunoglobulin were obtained from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA).
Cells and cell culture
The human ovarian carcinoma cell line 2008 that expresses both CLDN3 and CLDN4 and its CLDN3 knockdown subline 2008-CLDN3KD-4.5 [12] were grown in RPMI 1640 supplemented with 5% fetal bovine serum. Cultures were maintained at 37[degrees]C in a humidified atmosphere of 5% CO[sub.2 ]and 95% air.
Plasmid construction
The gene encoding gelonin in the …
No comments:
Post a Comment