Photochemical internalization of CD133-targeting immunotoxins efficiently depletes sarcoma cells with stem-like properties and reduces tumorigenicity

Background

The normal stem cell marker CD133 is also a putative marker of cancer stem cells (CSCs) in different types of cancers. Hence, a major challenge when targeting CD133-expressing CSCs is to prevent depletion of the normal stem cell pool. We hypothesized that the site-specific and light-controlled drug delivery method photochemical internalization (PCI) may have the potential to enhance selectivity and endosomal escape of CD133-targeting immunotoxins in stem-like sarcoma cells.

Methods

We have used a sarcoma model, SW872 cells isolated from xenografts harboring CSCs within a ~ 2% CD133^high subpopulation to investigate the potential of PCI of CD133-targeting toxin as a novel strategy to kill CSCs. Model immunotoxins were generated by binding the ribosome-inactivating protein toxin saporin to each of the monoclonal antibodies CD133/1 (AC133) or CD133/2 (293C), specific for individual CD133-epitopes. Cellular targeting, intracellular co-localization with the PCI photosensitizer, disulfonated meso-tetraphenylchlorin (TPCS_2a), and cytotoxic efficacy of PCI of the CD133-targeting toxins were evaluated.

Results

PCI of CD133–saporin efficiently targets CD133-expressing SW872 and HT1080 sarcoma cells and results in loss of cell viability. Following sub-toxic treatment, surviving SW872 cells, depleted of the CD133-expressing population, display reduced proliferative capacity and attenuated CSC properties, such as reduced colony-forming ability and tumorigenicity.

Conclusion

Here we present a proof-of-concept study, where PCI enables light-triggered delivery of CD133-targeting antibody-drug conjugates, resulting in decreased sarcoma tumor-initiating capacity.

General significance

PCI of CD133-targeting toxins may be used as a minimal invasive strategy in the treatment of sarcomas, and potentially as a therapeutic for other solid tumors expressing CD133.     

Variation in the Morphology of Bacillus mycoides Due to Applied Force and Substrate Structure

Response to mechanical force is a well characterised phenomenon in eukaryotic organisms, helping to organise multicellular structures. Mechanotactic responses have only rarely been observed in prokaryotic taxa. This work reports on a morphological change due to variations in applied force and surface structure by Bacillus mycoides Flügge. B. mycoides is a ubiquitous soil organism well known among microbiologists for its characteristic spreading colony morphology. An apparent mechanotactic response is elicited by physical deformation of the gel media on which B.mycoides is growing, including applied forces of compression or tension. Variations in the surface such as curvature produced by casting the agar gel in the presence of curved objects also elicited the change. The morphological change in B.mycoides colonies associated with the application of force manifests as a pattern of parallel rhizoid filaments perpendicular to compressing force and parallel to stretching force in the agar medium. The phenomenon is most clearly demonstrated by reversible changes in the orientation of B. mycoides filaments during time-lapse microscopy.     

Phosphorus release from sediments in a treatment wetland: Contrast between DET and EPC0 methodologies

Wetlands are capable of reducing nutrient loadings to receiving water bodies, and hence many artificial wetlands have been constructed for wastewater nutrient removal. In this study, diffusive equilibrium in thin films (DETs) and equilibrium phosphorus concentration (EPC₀) analysis were used to examine the role of sediment as a nutrient source or sink in a constructed treatment wetland in summer. The effect of dredging on sediment-water nutrient exchange was also studied. Soluble reactive phosphorus (SRP), ammonium (NH₄⁺) and sulphate (SO₄²⁻) concentration profiles were measured by DET across the sediment-water interface (SWI) in both a settling pond and iris reed bed within the wetland. The SRP concentrations in the sediment pore-waters of the settling pond were extremely high (up to 29,500 μg l⁻¹) near the SWI. This is over an order of magnitude higher than the levels found in the water column, which in turn are over an order of magnitude higher than environmental levels proposed to limit eutrophication in rivers. The profiles demonstrated an average net release of SRP and NH₄⁺ from the settling pond sediment to the overlying water of 58 mg m⁻² d⁻¹ (±32 mg m⁻² d⁻¹ (1 sd)) and 16 mg m⁻² d⁻¹ (±25 mg m⁻² d⁻¹ (1 sd)), respectively. The DET SO₄²⁻ concentration profiles revealed that the sediment was anoxic within 2 cm of the SWI. Dredging of the reed bed made no significant difference to the P release characteristics across the SWI. The EPC₀s were much lower than the SRP concentration of the overlying water, indicating that the sediment had the potential to act as a phosphate sink. The apparent contradiction of the DET and EPC₀ results is attributed to the fact that DET measurements are made in situ, where as EPC₀ measurements are ex situ. These results show that substantial releases of P can occur from wetland sediments, and also highlight the need for caution when interpreting ex situ EPC₀ analytical results.     

Hypoxia-driven elimination of thiopurines from their nitrobenzyl prodrugs

A novel bioreductive prodrug of 6-thioguanine, 2-amino-6-[2-(4-nitrophenyl)prop-2-ylsulfanyl]-9H-purine, containing a gem-dimethyl thioether linkage, was synthesised and compared with its unsubstituted analogue. In A549 whole cell experiments hypoxia selective release of 6-thioguanine was observed with the substituted prodrug only.     

Arene cis-dihydrodiols: useful precursors for the preparation of analogues of the anti-tumour agent, 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC)

The synthesis of 6-epi-COTC, a diastereoisomer of Streptomyces metabolite 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC), is described. The anti-cancer activities of the novel analogue, in racemic and enantiomerically pure forms, are presented.     

Analogues of 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC) with anti-tumor properties

The syntheses of three novel analogues of the naturally occurring cytotoxic agent COTC are described and the results of bioassays of the target compounds against two lung cancer cell lines are presented.     

The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation,leading to G1 arrest

Cellular stresses, including growth factor deprivation, inflammatory cytokines or viral infection promote RAX/PACT-dependent activation of the double-stranded RNA-dependent protein kinase, PKR, to phosphorylate eIF2α, resulting in translation inhibition and apoptosis. In addition, PKR has been reported to regulate p53, STAT1 and NFκB. Here, we report that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while the expression of RAX/PACT, PKR or p53 alone has little effect on the cell cycle of p53-null H1299 cells, co-expression of p53 with either RAX/PACT or PKR promotes a 25–35% increase of cells in G₁. In contrast, co-expression of RAX/PACT with the sumoylation-deficient p53(K386R) mutant or with the desumoylase SENP1 fails to induce such a G₁ arrest. Furthermore, co-expression of p53, RAX/PACT and the dominant-negative PKR(K296R) mutant inhibits RAX/PACT-induced, p53-dependent G₁ growth arrest and expression of RAX/PACT in pkr^+/+ but not pkr^−/− MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment, and expression of exogenous RAX/PACT promotes phosphorylation of wild-type but not p53(K386R) on serine 392. Collectively, results indicate that, in response to stress, the RAX/PACT-PKR signaling pathway may inhibit p53 protein turnover by a sumoylation-dependent mechanism with promotion of p53 phosphorylation and translational activation leading to G₁ cell cycle arrest.Key words: p53, PKR, RAX, PACT, Ubc9, sumoylation     

Parentage analysis in a managed free ranging population of southern white rhinoceros: genetic diversity,pedigrees and management

Small populations are vulnerable to the consequences of breeding within closed groups as the loss of genetic variability can lead to inbreeding depression. Here, we use microsatellite genotypes to assess variability and parentage within a small, managed population of southern white rhinoceros in northern Namibia. Tissue samples gathered from either a modified biopsy darting technique or ear notches allowed us to obtain genotypic data for all individuals in the population. As expected for this species, genetic variability in the population was relatively low (overall H _obs 0.45). In combination with detailed management records for the period 1993–2009, we were able to assign both parents for all 23 offspring. Only one calf of seven in the F₂ generation arose from father–daughter inbreeding within the population. Our analysis revealed that paternity was initially dominated by a single founder bull siring 10 of 13 calves over 9 years; paradoxically, the other founder bull was selected for removal based on observations suggesting he was behaviourally dominant and therefore the likely sire of most calves. We also found that young introduced bulls were breeding successfully within 6 months of their arrival, well before having established their home ranges. We argue that in order to optimally manage and conserve the southern African white rhinoceros meta-population it is essential to have accurate pedigree information and genetic data for all individuals in the numerous small populations that are key to the survival of the species.     

Differential gene expression between African American and European American colorectal cancer patients

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations.     

Synthesis and enzymatic evaluation of pyridinium-substituted uracil derivatives as novel inhibitors of thymidine phosphorylase

A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil.