Factors implicated in the assessment of aminolevulinic acid-induced protoporphyrin IX fluorescence

Background

Photodynamic therapy and photodiagnosis of cancer requires preferential accumulation of fluorescent photosensitizers in tumors. Clinical evidence documents feasibility of ALA-based photodiagnosis for tumor detection. However, false positive results and large variations in fluorescence intensities are also reported. Furthermore, selective accumulation of fluorescent species of photosensitizers in tumor cell lines, as compared to normal ones, when cultured in vitro, is not always observed. To understand this discrepancy we analyzed the impact of various factors on the intensity of detected PpIX fluorescence.

Methods

Impacts of cell type, mitochondrial potential, cell–cell interactions and relocalization of PpIX among different cell types in co-cultures of different cell lines were analyzed by confocal microscopy and flow cytometry. Fluorescence spectroscopy was used to estimate absolute amounts of ALA-induced PpIX in individual cell lines. Immunofluorescence staining was applied to evaluate the ability of cell lines to produce collagen.

Results

Higher ALA-induced PpIX fluorescence in cancer cell lines as compared to normal ones was not detected by all the methods used. Mitochondrial activity was heterogeneous throughout the cell monolayers and could not be clearly correlated with PpIX fluorescence. Positive collagen staining was detected in all cell lines tested.

Conclusions

Contrary to in vivo situation, ALA-induced PpIX production by cell lines in vitro may not result in higher PpIX fluorescence signals in tumor cells than in normal ones. We suggest that a combination of several properties of tumor tissue, instead of tumor cells only, is responsible for increased ALA-induced PpIX fluorescence in solid tumors.

General significance

Understanding the reasons of increased ALA-induced PpIX fluorescence in tumors is necessary for reliable ALA-based photodiagnosis, which is used in various oncological fields.     

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.     

Synthesis and toxicity characterization of carbon coated iron oxide nanoparticles with highly defined size distributions

Background

Iron oxide nanoparticles hold great promise for future biomedical applications. To this end numerous studies on iron oxide nanoparticles have been conducted. One aspect these studies reveal is that nanoparticle size and shape can trigger different cellular responses through endocytic pathways, cell viability and early apoptosis. However, systematic studies investigating the size dependence of iron oxide nanoparticles with highly defined diameters across multiple cells lines are not available yet.

Methods

Iron oxide nanoparticles with well-defined size distributions were prepared. All samples were thoroughly characterized and the cytotoxicity for four standard cell lines (HeLa Kyoto, human osteosarcoma (U2OS), mouse fibroblasts (NIH 3T3) and mouse macrophages (J7442)) where investigated.

Results

Our findings show that small differences in size distribution (ca. 10 nm) of iron oxide nanoparticles do not influence cytotoxicity, while uptake is size dependent. Cytotoxicity is dose-dependent. Broad distributions of nanoparticles are more easily internalized as compared to the narrow distributions for two of the cell lines tested (HeLa Kyoto and mouse macrophages (J7442)).

Conclusion

The data indicate that it is not feasible to probe changes in cytotoxicity within a small size range (10 nm). However, TEM investigations of the nanoparticles indicate that cellular uptake is size dependent.

General significance

The present work compares narrow and broad distributions for various samples of carbon-coated iron oxide nanoparticles. The data highlights that cells differentiate between nanoparticle sizes as indicated by differences in cellular uptake. This information provides valuable knowledge to better understand the interaction of nanoparticles and cells.     

Effect of the Photodynamic Therapy Applications with Potent Microalgae Constituents on Several Types of Tumor

Background

In recent years, microalgae (MA) have attracted much interest considering their possible therapeutic application. They contain active natural compounds or derivatives (extracts, pure or chemically modified compounds) that have increasing applications in the pharmaceutical industry.

Glycosaminoglycans and syndecan-4 are involved in SDF-1/CXCL12-mediated invasion of human epitheloid carcinoma HeLa cells

Background

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 mediates its biological activities through activation of G protein-coupled receptor CXCR4 and binds to glycosaminoglycans (GAGs).

Methods

Using Bio-coat cell migration chambers, specific antagonists, flow cytometry and RNA interference, we evaluate the involvement of heparan sulfate proteoglycans (HSPG) in the SDF-1/CXCL12-induced invasion of human cervix epitheloid carcinoma HeLa cells.

Results

The SDF-1/CXCL12-induced cell invasion is dependent on CXCR4. Furthermore, Protein Kinase C delta (PKC δ) and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) are implicated in this event, but not extracellular signal-regulated kinase (ERK) 1/2. Moreover, the invasion of HeLa cells induced by SDF-1/CXCL12 was dependent on matrix metalloproteinase-9 (MMP-9). The pre-incubation of HeLa cells with heparin or with anti-heparan sulfate antibodies or with β-d-xyloside inhibited SDF-1/CXCL12-mediated cell invasion. Furthermore, the down-regulation of syndecan-4, a heparan sulfate proteoglycan, decreased SDF-1/CXCL12-mediated HeLa cell invasion. GAGs, probably on syndecan-4, are involved in SDF-1/CXCL12-mediated cell chemotaxis.

General significance

These data suggest that targeting the glycosaminoglycan/chemokine interaction could be a new therapeutic approach for carcinomas in which SDF-1/CXCL12 is involved.     

Monitoring and manipulation of the pH of single cells using infrared spectromicroscopy and a molecular switch

Background

The pH of a biological system is a crucial determinant of the structures and reactivity of its components and cellular homeostasis of H⁺ is critical for cell viability. Control and monitoring of cellular acidity are highly desirable for the purpose of studying biochemical processes in vivo.

Methods

The effect of photolysis of a caged strong acid, the ester 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) is used to cause a controlled drop in pH in single cells. An isolated cell is selected under the IR microscope, irradiated with near-UV light and monitored by FTIR.

Results

We demonstrate the use of FTIR spectromicroscopy to monitor light-induced acidification of the cellular medium by measuring the increased concentration of CO₂ and corresponding decrease of HCO₃⁻ in the cell and in the surrounding medium.

Conclusions

We have demonstrated a method to control and accurately monitor the changes in pH of a cellular system by coupling a caged proton-releasing agent with FTIR spectromicroscopy detection. The overall implementation of photolysis and spectroscopic detection in a microscope optical configuration ensures single cell selectivity in both acidification and monitoring. We show the viability of monitoring of pH changes by FTIR spectromicroscopy with sensitivity comparable to that of glass electrodes, better than the existing methods for determining cell pH.

General significance

Reporting the effect of small variations of cellular acidity provides a major improvement in the understanding of the interplay between molecular properties as assessed in vitro and cell physiology.     

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound,inhibits human oral squamous carcinoma cells KB and KB/VCR: In vitro and in vivo studies

Background

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, might possess anti-cancer properties. We aimed to evaluate the efficacy of Riccardin D-26 as a candidate compound for treatment of cancers with sensitive or drug resistant cells.

Methods

Experiments were performed on human oral squamous carcinoma KB cells and vincristin-selected MDR KB/VCR cells. The inhibition of cell growth was evaluated by colorimetric and clonogenic assays. The apoptotic cells were determined by the Annexin V-FITC/PI staining assay. JC-1 fluorescence probe was used to examine the mitochondria membrane potential (MMP). Further experiments were performed in nude mice bearing KB or KB/VCR xenografts. Riccardin D-26 was administered by injection for 2 weeks. The specimens of KB and KB/VCR xenografts were removed for TUNEL staining and Western blotting analysis.

Results

Riccardin D-26 significantly inhibited cancer growth in both KB and KB/VCR cells. Riccardin D-26's activity in cancer cells was greater than that in human normal liver cells. In mice, Riccardin D-26 effectively prevented the growth of KB and KB/VCR xenografts without significant toxicity. Further studies suggested that Riccardin D-26 inhibited cancer growth by inducing apoptosis in the activation of mitochondria-mediated intrinsic apoptosis pathway. Riccardin D-26 also possessed this activity in regulation of mitogen-related protein kinases such as MAPK and PI3K/Akt, which is associated with its inhibitory effect on KB/VCR cells.

Conclusions

Riccardin D-26 possessed an anti-proliferation activity against both sensitive KB and MDR KB/VCR cancer cells.

General significance

Riccardin D-26 could be a promising agent for treatment of cancers with sensitive or drug resistant cells.     

Extensive evaluations of the cytotoxic effects of gold nanoparticles

Background

Many in vitro studies have revealed that the interference of dye molecules in traditional nanoparticle cytotoxicity assays results in controversial conclusions. The aim of this study is to establish an extensive and systematic method for evaluating biological effects of gold nanoparticles in mammalian cell lines.

Methods

We establish the cell-impedance measurement system, a label-free, real-time cell monitoring platform that measures electrical impedance, displaying results as cell index values, in a variety of mammalian cell lines. Cytotoxic effects of gold nanoparticles are also evaluated with traditional in vitro assays.

Results

Among the six cell lines, gold nanoparticles induce a dose-dependent suppression of cell growth with different levels of severity and the suppressive effect of gold nanoparticles was indirectly associated with their sizes and cellular uptake. Mechanistic studies revealed that the action of gold nanoparticles is mediated by apoptosis induction or cell cycle delay, depending on cell type and cellular context. Although redox signaling is often linked to the toxicity of nanoparticles, in this study, we found that gold nanoparticle-mediated reactive oxygen species generation was not sustained to notably modulate proteins involved in antioxidative defense system.

Conclusion

The cell-impedance measurement system, a dye-free, real-time screening platform, provides a reliable analysis for monitoring gold nanoparticle cytotoxicity in a variety of mammalian cell lines. Furthermore, gold nanoparticles induce cellular signaling and several sets of gene expression to modulate cellular physical processes.

General significance

The systematic approach, such as cell-impedance measurement, analyzing the toxicology of nanomaterials offers convincing evidence of the cytotoxicity of gold nanomaterials.     

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

Background

The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.

Methods

Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.

Results

m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.

Conclusions

Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.

General significance

These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.     

Hydogen peroxide-dependent photocytotoxicity by phloxine B,a xanthene-type food colorant

Background

Phloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent.

Methods

We investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants.

Reuslts and conclusions

PhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H₂O₂), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H₂O₂ in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H₂O₂-dependent mechanism.

General significance

Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.     

SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner

Aims

The mechanism by which SR48692 inhibits non-small cell lung cancer (NSCLC) proliferation was investigated.

Main methods

The ability of SR48692 to inhibit the proliferation of NSCLC cell lines NCI-H1299 and A549 was investigated in vitro in the presence or absence of neurotensin (NTS). The ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation was investigated by Western blot using NSCLC cells and various inhibitors. The growth effects and Western blot results were determined in cell lines treated with siRNA for NTSR1.

Key findings

Treatment of A549 or NCI-H1299 cells with siRNA for NTSR1 reduced significantly NTSR1 protein and the ability of SR48692 to inhibit the proliferation of A549 or NCI-H1299 NSCLC cells. Treatment of A549 and NCI-H1299 cells with siRNA for NTSR1 reduced the ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation. SR48692 or gefitinib (EGFR tyrosine kinase inhibitor) inhibited the ability of NTS to cause EGFR and ERK tyrosine phosphorylation. NTS transactivation of the EGFR was inhibited by GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) or U73122 (phospholipase C inhibitor) but not H89 (PKA inhibitor). NTS stimulates whereas SR48692 or gefitinib inhibits the clonal growth of NSCLC cells.

Significance

These results suggest that SR48692 may inhibit NSCLC proliferation in an EGFR-dependent mechanism.     

Regulation of post-translational protein arginine methylation during HeLa cell cycle

Background

Post-translational arginine methylation which modifies protein-arginyl residues by protein arginine methyltransferase (PRMT) was investigated during synchronized HeLa cell cycle.

Methods

The lysates of cells synchronized at each stage were subjected to one and/or two dimensional electrophoresis followed by Western immunoblot using against anti-asymmetric-dimethyl-arginine (ASYM24), anti-symmetric-dimethyl-arginine (SYM10), and subclasses of PRMTs, including PRMT1, PRMT3, PRMT4 (CARM1), PRMT5, PRMT6, and PRMT7 antibodies.

Results

Proteins with approximate molecular masses of 80 kDa, 68 kDa, and 64 kDa, containing asymmetric-dimethyl-arginine (aDMA) were increased at G0/G1 to G1, which lasted until S phase. In addition, 25 kDa protein of symmetric-dimethyl-arginine (sDMA) was also markedly up-regulated from G0/G1 to G1. The levels of PRMT3, PRMT6 and PRMT7 were concurrently increased during the cell cycle. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS was identified as aDMA-80 kDa and aDMA-68 kDa proteins as heterogeneous nuclear ribonucleoprotein R (hnRNPR), aDMA-64 kDa proteins as cleavage stimulation factor 64 kDa subunit (CstF-64), and sDMA-25 kDa protein as triosephosphate isomerase (TPI). The levels of increased aDMA of hnRNPR were reduced, when HeLa cells were transfected with siRNA for PRMT1, and the aDMA of CstF-64 with siRNA for PRMT3, while depletion of PRMT5 down-regulated sDMA of TPI.

Conclusion

Protein arginine dimethylations of hnRNPR, CstF-64, and TPI were regulated during HeLa cell cycle by respective PRMTs.

General significance

These results suggest that regulation of arginine dimethylation of hnRNPR, CstF-64, and TPI at G0/G1 to G1 are most likely to modulate the cellular growth and proliferation in HeLa cell cycle.     

Effects of celastrol on human cervical cancer cells as revealed by ion-trap gas chromatography–mass spectrometry based metabolic profiling

Background

Celastrol, a quinine methide triterpene extracted from a Chinese medicine (Trypterygium wilfordii Hook F.), has the potential to become an anticancer drug with promising prospects. Cell culture metabolomics has been a powerful method to study metabolic profiles in cell line after drug treatment, which can be used for discovery of drug targets and investigation of drug effects.

Methods

We analyzed the metabolic modifications induced by celastrol treatment in human cervical cancer cells, using an ion-trap gas chromatography–mass spectrometry based metabolomics combined with multivariate statistical analysis, which allows simultaneous screening of multiple characteristic metabolic pathways related to celastrol treatment. Three representative apoptosis-inducing cytotoxic agents, namely cisplatin, doxorubicin hydrochloride and paclitaxel, were selected as positive control drugs to validate reasonableness and accuracy of our metabolomic investigation on celastrol.

Results

Anti-proliferation and apoptotic effects of celastrol were demonstrated by CCK-8 assay, Annexin-V/PI staining method, mitochondrial membrane potential (ΔΨm) assay and caspase-3 assay. Several significant metabolites involved in energy, amino acid and nucleic acid metabolism in HeLa cells induced by celastrol and positive drugs were reported. Our method is proved to be effective and robust to provide new evidence of pharmacological mechanism of celastrol.

Conclusions

The metabolic alterations induced by drug treatment showed the impaired physiological activity of HeLa cells, which also indicated anti-proliferative and apoptotic effects of celastrol and these positive drugs.

General significance

GC/MS-based metabolomic approach applied to cell culture could give valuable information on the systemic effects of celastrol in vitro and help us to further study its anticancer mechanism.     

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease

Background

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.

Methods

Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.

Results

ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.

Conclusions

ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.

General significance

The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.     

The TGFβ superfamily in stem cell biology and early mammalian embryonic development

Background

Members of the Transforming Growth Factor-beta (TGFβ) superfamily of cytokines are essential for early embryonic development and play crucial roles in pluripotency and differentiation of embryonic stem cells in vitro.

Scope of review

In this review, we discuss how TGFβ family signals are read by cells and how they are modulated by the cellular context. Furthermore, we review recent advances in our understanding of TGFβ function in embryonic stem cells and point out hot topics at the intersection of TGFβ signaling and stem cell biology fields.

Major conclusion

TGFβ family signals are essential for early mammalian development and the importance of this pathway is reflected in pluripotent stem cells derived from the mammalian embryo.

General significance

Understanding signaling pathways underlying pluripotency and cell fate specification holds promises for the advent of personalized regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

A nuclear ligand MRG15 involved in the proapoptotic activity of medicinal fungal galectin AAL (Agrocybe aegerita lectin)

Background

We have previously reported a novel fungal galectin Agrocybe aegerita lectin (AAL) with apoptosis-induced activity and nuclear migration activity. The importance of nuclear localization for AAL's apoptosis-induced activity has been established by mutant study. However, the mechanism remains unclear.

Methods

We further investigated the mechanism using a previously reported carbohydrate recognition domain (CRD) mutant protein H59Q, which retained its nuclear localization activity but lost most of its apoptotic activity. The cell membrane-binding ability of recombinant AAL (rAAL) and H59Q was analyzed by FACS, and their cellular partners were identified by affinity chromatography and mass spectroscopy. Furthermore, the interaction of AAL and ligand was proved by mammalian two-hybrid and pull down assays. A knockdown assay was used to confirm the role of the ligand.

Results

The apoptotic activity of AAL could be blocked by lactose. Mutant H59Q retained comparable cell membrane-binding ability to rAAL. Four cellular binding partners of AAL in HeLa cells were identified: glucose-regulated protein 78 (GRP78); mortality factor 4-like protein 1 (MRG15); elongation factor 2 (EEF2); and heat shock protein 70 (Hsp70). CRD region of AAL was required for the interaction between AAL/mutant AAL and MRG15. MRG15 knockdown increased the cells' resistance to AAL treatment.

Conclusion

MRG15 was a nuclear ligand for AAL in HeLa cells. These data implied the existence of a novel nuclear pathway for the antitumor activity of fungal galectin AAL.

General significance

These findings provide a novel explanation of AAL bioactivity and contribute to the understanding of mushroom lectins' antitumor activity.     

Ubiquitination and SUMOylation of annexin A1 and helicase activity

Background

While annexin A1 in nuclei is proposed to be involved in cell transformation, its functions remain poorly understood. Since annexin A1 has the consensus motif, ¹⁶⁰LKRD, for SUMOylation as well as Ks, acceptors for ubiquitination that regulates localization and functions of proteins, we investigated SUMOylation and ubiquitination of annexin A1.

Methods

SUMOylation and ubiquitination of bovine annexin A1 were biochemically tested in vitro by purified proteins, and were confirmed by cell experiments with L5178 lymphoma cells. Effects of the modifications on DNA helicase activity were measured by ssDNA binding activity and by dsDNA unwinding activity.

Results

SUMOylation of annexin A1 was catalyzed by Ubc9, while its ubiquitination was by Rad6-Rad 18. Ubiquitinated annexin A1 had higher affinity for damaged DNA, and promoted in vitro translesion DNA synthesis by Pol β. In mouse lymphoma L5178Y tk(+/−) cells, levels of SUMOylated annexin A1 decreased by DNA damaging agents, MMS or As³⁺, whereas those of ubiquitinated annexin A1 increased under the same conditions.

Conclusion

These observations suggest but do not necessarily prove that ubiquitinated annexin A1 in nuclei may be involved in DNA damage response, while SUMOylated annexin A1 functions in proliferation–differentiation.

Significance

Ubiquitination of annexin A1 may play an important role in mutagenesis, an initial step of cell transformation.     

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.