Riproximin Exhibits Diversity in Sugar Binding, and Modulates some Metastasis-Related Proteins with Lectin like Properties in Pancreatic Ductal Adenocarcinoma

Abstract

Riproximin (Rpx) is a type II ribosome-inactivating protein with specific anti-proliferative activity. It was purified from Ximenia americana by affinity chromatography using a resin coupled with lactosyl residues. The same technique facilitated isolation of proteins with lectin-like properties from human Suit2-007 and rat ASML pancreatic cancer cells, which were termed lactosyl-sepharose binding proteins (LSBPs). The role of these proteins in cancer progression was investigated at mRNA level using chip array data of Suit2-007 and ASML cells re-isolated from nude rats. These data compared significant mRNA expression changes when relating primary (pancreas) and metastatic (liver) sites following orthotopic and intraportal implantation of Pancreatic Ductal Adenocarcinoma (PDAC) cells, respectively. The affinity of Rpx to 13 simple sugar structures was modeled by docking experiments, the ranking of which was principally confirmed by NMR-spectroscopy. In addition, Rpx and LSBPs were evaluated for anti-proliferative activity and their cellular uptake was assessed by fluorescence microscopy. From 13 monosaccharides evaluated, open-chain rhamnose, β-d-galactose, and α-l-galactopyranose showed the highest affinities for site 1 of Rpx’s B-chain. NMR evaluation yielded a similar ranking, as galactose was among the best binders. Both, Rpx and LSBPs reduced cell proliferation in vitro, but their anti-proliferative effects were decreased by 15–20% in the presence of galactose. The program “Ingenuity Pathway Analysis” identified 2,415 genes showing significantly modulated mRNA expression following exposure of Suit2-007 cells to Rpx in vitro. These genes were then matched to those 1,639 genes, which were significantly modulated in the rat model when comparing primary and metastatic growth of Suit2-007 cells. In this overlap analysis, LSBP genes were considered separately. The potential suitability of Rpx for treating metastatic Suit2-007 PDAC cells was reflected by those genes, which were modulated by Rpx in a way opposite to that observed in cancer progression. Remarkably, these were 14% of all genes modulated during cancer progression, but 71% of the respective LSBP gene subgroup. Based on these findings, we predict that Rpx has the potential to treat PDAC metastasis by modulating genes involved in metastatic progression, especially by targeting LSBPs.

Khasianine Affects the Expression of Sugar-Sensitive Proteins in Pancreatic Cancer Cells, Which Are Altered in Data from the Rat Model and Patients

“…The first set was derived from Suit2-007 cells re-isolated from tumors growing in the liver environment of nude rats after intraportal implantation into this organ . The second set was chip array derived from ASML cells growing at different stages of liver colonization in the immuno­competent BDX rat. , The third set was from pancreatic cancer patients (tumor versus normal tissue) downloaded from the GEO (ID: GSE71989) data base …”

“…Total RNA was isolated as detailed in the Fast Gene RNA isolation kit (Nippon Genetics Co. Ltd.). The concentrations of total RNA in samples were determined by a Nanodrop spectrophotometer and chip array performed according to the modified Eberwine protocol. , …”

“…In our previous study, we demonstrated that PDAC cell lines (Suit2-007 and ASML) express lactosyl-Sepharose binding proteins (LSBPs), which were shown to bind simple sugars, including galactose (Gal), glucose (Glc), fucose (Fuc), mannose (Man) and rhamnose (Rha). − It was also evident that only a subgroup of these proteins could bind these sugars. As some of these proteins were significantly expressed in a rat model for liver metastasis and in data from pancreatic cancer patients, it was anticipated that this property (sugar binding) could offer a rationale for evaluating the biological activity of sugar-bearing compounds against PDAC.…”

“…8,9 These regimens, however, confer only marginal benefits to patients and are often associated with side effects. 10 Therefore, novel drugs with less or, preferably, no side effects, which can prolong patient survival, are urgently needed.…”

Anticancer genes (NOXA, PAR-4, TRAIL) are de-regulated in breast cancer patients and can be targeted by using a ribosomal inactivating plant protein (riproximin)

“…As far as the molecular structure is concerned, riproximin is a heterodimer with two polypeptide chains (A-and B-chains) held together by an intermolecular disulphide bridge. The B-chain of riproximin has lectin-like properties and is responsible for binding to cell surface glycans, while the A-chain is accountable for subsequent catalytic activity [20,21]. Riproximin has demonstrated substantial antineoplastic effects against a variety of cancer cell lines including breast, colorectal, leukaemia, pancreatic and prostate, while sparing normal healthy cells [22][23][24][25][26].…”