Cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles: toxicity and changes in the proteome of breast, colon and prostate cancer cells.

Nanoparticles composed of poly(alkyl cyanoacrylate) (PACA) have shown great promise due to their biodegradability and high drug loading capacity. Development of optimal PACA nanocarriers requires detailed analysis of the overall cellular impact exerted by PACA variants. We here perform a comprehensive comparison of cabazitaxel (CBZ)-loaded nanocarriers composed of three different PACA monomers, i.e. poly(n-butyl cyanoacrylate) (PBCA), poly(2-ethylbutyl cyanoacrylate) (PEBCA) and poly(octyl cyanoacrylate) (POCA). The cytotoxicity of drug-loaded and empty PACA nanoparticles were compared to that of free CBZ across a panel of nine cancer cell lines by assessing cellular metabolism, proliferation and protein synthesis. The analyses revealed that the cytotoxicity of all CBZ-loaded PACAs was similar to that of free CBZ for all cell lines tested, whereas the empty PACAs exerted much lower toxicity. To increase our understanding of the toxic effects of these treatments comprehensive MS-based proteomics were performed with HCT116, MDA-MB-231 and PC3 cells incubated with PACA-CBZ variants or free CBZ. Interestingly, PACA-CBZ specifically led to decreased levels of proteins involved in focal adhesion and stress fibers in all cell lines. Since we recently demonstrated that encapsulation of CBZ within PEBCA nanoparticles significantly improved the therapeutic effect of CBZ on a patient derived xenograft model in mice, we investigated the effects of this PACA variant more closely by immunoblotting. Interestingly, we detected several changes in the protein expression and degree of phosphorylation of SRC-pathway proteins that can be relevant for the therapeutic effects of these substances.

Drug-Loaded Photosensitizer-Chitosan Nanoparticles for Combinatorial Chemo- and Photodynamic-Therapy of Cancer.

In this study we have developed biodegradable polymeric nanoparticles (NPs) containing the cytostatic drugs mertansine (MRT) or cabazitaxel (CBZ). The NPs are based on chitosan (CS) conjugate polymers synthesized with different amounts of the photosensitizer tetraphenylchlorin (TPC). These TPC-CS NPs have high loading capacity and strong drug retention due to π-π stacking interactions between the drugs and the aromatic photosensitizer groups of the polymers. CS polymers with 10% of the side chains containing TPC were found to be optimal in terms of drug loading capacity and NP stability. The TPC-CS NPs loaded with MRT or CBZ displayed higher cytotoxicity than the free form of these drugs in the breast cancer cell lines MDA-MB-231 and MDA-MB-468. Furthermore, light-induced photochemical activation of the NPs elicited a strong photodynamic therapy effect on these breast cancer cells. Biodistribution studies in mice showed that most of the TPC-CS NPs accumulated in liver and lungs, but they were also found to be localized in tumors derived from HCT-116 cells. These data suggest that the drug-loaded TPC-CS NPs have a potential in combinatory anticancer therapy and as contrast agents.

Small variations in nanoparticle structure dictate differential cellular stress responses and mode of cell death.

For optimal exploitation of nanoparticles (NPs) in biomedicine, and to predict nanotoxicity, detailed knowledge of the cellular responses to cell-bound or internalized NPs is imperative. The final outcome of NP-cell interaction is dictated by the type and magnitude of the NP insult and the cellular response. Here, this has been systematically studied by using poly(alkylcyanoacrylate) (PACA) particles differing only in their alkyl side chains; butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), respectively. Surprisingly, these highly similar NPs induced different stress responses and modes of cell death in human cell lines. The POCA particles generally induced endoplasmic reticulum stress and apoptosis. In contrast, PBCA and PEBCA particles induced oxidative stress and lipid peroxidation depending on the level of the glutathione precursor cystine and transcription of the cystine transporter SLC7A11. The latter was induced as a protective response by the transcription factors ATF4 and Nrf2. PBCA particles strongly activated ATF4 downstream of the eIF2α kinase HRI, whereas PEBCA particles more potently induced Nrf2 antioxidant responses. Intriguingly, PBCA particles activated the cell death mechanism ferroptosis; a promising option for targeting multidrug-resistant cancers. Our findings highlight that even minor differences in NP composition can severely impact the cellular response to NPs. This may have important implications in therapeutic settings.

Cabazitaxel-loaded Poly(2-ethylbutyl cyanoacrylate) nanoparticles improve treatment efficacy in a patient derived breast cancer xenograft.

The effect of poly(2-ethyl-butyl cyanoacrylate) nanoparticles containing the cytotoxic drug cabazitaxel was studied in three breast cancer cell lines and one basal-like patient-derived xenograft model grown in the mammary fat pad of immunodeficient mice. Nanoparticle-encapsulated cabazitaxel had a much better efficacy than similar concentrations of free drug in the basal-like patient-derived xenograft and resulted in complete remission of 6 out of 8 tumors, whereas free drug gave complete remission only with 2 out of 9 tumors. To investigate the different efficacies obtained with nanoparticle-encapsulated versus free cabazitaxel, mass spectrometry quantification of cabazitaxel was performed in mice plasma and selected tissue samples. Nanoparticle-encapsulated drug had a longer circulation time in blood. There was approximately a three times higher drug concentration in tumor tissue 24 h after injection, and two times higher 96 h after injection of nanoparticles with drug compared to the free drug. The tissue biodistribution obtained after 24 h using mass spectrometry analyses correlates well with biodistribution data obtained using IVIS® Spectrum in vivo imaging of nanoparticles labeled with the fluorescent substance NR668, indicating that these data also are representative for the nanoparticle distribution. Furthermore, immunohistochemistry was used to estimate infiltration of macrophages into the tumor tissue following injection of nanoparticle-encapsulated and free cabazitaxel. The higher infiltration of anti-tumorigenic versus pro-tumorigenic macrophages in tumors treated with the nanoparticles might also contribute to the improved effect obtained with the nanoparticle-encapsulated drug. Tumor infiltration of pro-tumorigenic macrophages was four times lower when using nanoparticles containing cabazitaxel than when using particles without drug, and we speculate that the very good therapeutic efficacy obtained with our cabazitaxel-containing particles may be due to their ability to reduce the level of pro-tumorigenic macrophages in the tumor. In summary, encapsulation of cabazitaxel in poly(2-ethyl-butyl cyanoacrylate) nanoparticles seems promising for treatment of breast cancer.

Ceramide-containing liposomes with doxorubicin: time and cell-dependent effect of C6 and C12 ceramide.

Doxorubicin, a widely used chemotherapeutic drug, has several potential high-risk side effects including cardiomyopathy. Furthermore, cellular resistance to this drug develops with time. By using liposomes as carrier vesicles both the side effects and drug resistance might be avoided. In this study we have investigated the cytotoxic effect of doxorubicin encapsulated in liposomes with and without ceramides containing 6 or 12 carbon atoms in the N-amidated fatty acyl chains. The short-chain ceramide species were included in the liposomal compositions due to their pro-apoptotic properties, which might cause a synergistic anticancer effect. We demonstrate that the ceramide species enhance the liposomal doxorubicin toxicity in a cell-specific manner. The C6-ceramide effect is most pronounced in cervical cancer cells (HeLa) and colon cancer cells (HCT116), whereas the C12-ceramide effect is strongest in breast cancer cells (MDA-MB-231). Moreover, the study reveals the importance of investigating cell toxicity at several time points and in different cell-lines, to assess drug-and formulation-induced cytotoxic effects in vitro. Furthermore, our data show that the cytotoxicity obtained with the nanocarriers in vitro, does not necessarily reflect their ability to inhibit tumor growth in vivo. We speculate that the larger effect of Caelyx® than our liposomes in vivo is due to a greater in vivo stability of Caelyx®.

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

PIKfyve inhibition increases exosome release and induces secretory autophagy.

Exosomes are vesicles released from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane. This study aimed to investigate whether the phosphoinositide kinase PIKfyve affects this process. Our results show that in PC-3 cells inhibition of PIKfyve by apilimod or depletion by siRNA increased the secretion of the exosomal fraction. Moreover, quantitative electron microscopy analysis showed that cells treated with apilimod contained more MVBs per cell and more intraluminal vesicles per MVB. Interestingly, mass spectrometry analysis revealed a considerable enrichment of autophagy-related proteins (NBR1, p62, LC3, WIPI2) in exosomal fractions released by apilimod-treated cells, a result that was confirmed by immunoblotting. When the exosome preparations were investigated by electron microscopy a small population of p62-labelled electron dense structures was observed together with CD63-containing exosomes. The p62-positive structures were found in less dense fractions than exosomes in density gradients. Inside the cells, p62 and CD63 were found in the same MVB-like organelles. Finally, both the degradation of EGF and long-lived proteins were shown to be reduced by apilimod. In conclusion, inhibition of PIKfyve increases secretion of exosomes and induces secretory autophagy, showing that these pathways are closely linked. We suggest this is due to impaired fusion of lysosomes with both MVBs and autophagosomes, and possibly increased fusion of MVBs with autophagosomes, and that the cells respond by secreting the content of these organelles to maintain cellular homeostasis.

Identification of prostate cancer biomarkers in urinary exosomes.

Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-specific molecules can be found in exosomes isolated from biological fluids. We have here investigated the proteome of urinary exosomes by using mass spectrometry to identify proteins differentially expressed in prostate cancer patients compared to healthy male controls. In total, 15 control and 16 prostate cancer samples of urinary exosomes were analyzed. Importantly, 246 proteins were differentially expressed in the two groups. The majority of these proteins (221) were up-regulated in exosomes from prostate cancer patients. These proteins were analyzed according to specific criteria to create a focus list that contained 37 proteins. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. Even though several of these proteins showed high sensitivity and specificity for prostate cancer as individual biomarkers, combining them in a multi-panel test has the potential for full differentiation of prostate cancer from non-disease controls. The highest sensitivity, 94%, was observed for transmembrane protein 256 (TM256; chromosome 17 open reading frame 61). LAMTOR proteins were also distinctly enriched with very high specificity for patient samples. TM256 and LAMTOR1 could be used to augment the sensitivity to 100%. Other prominent proteins were V-type proton ATPase 16 kDa proteolipid subunit (VATL), adipogenesis regulatory factor (ADIRF), and several Rab-class members and proteasomal proteins. In conclusion, this study clearly shows the potential of using urinary exosomes in the diagnosis and clinical management of prostate cancer.

Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs.

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

The ether lipid precursor hexadecylglycerol stimulates the release and changes the composition of exosomes derived from PC-3 cells.

Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes.

Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain.

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.

Sequestration assays for mammalian autophagy.

Macroautophagic activity is most directly and precisely measured by a cargo sequestration assay. Long-lived, cytosolic proteins that are degraded exclusively by the autophagic-lysosomal pathway, such as lactate dehydrogenase (LDH) are suitable as endogenous sequestration probes. Autophagic sequestration is measured as transfer of the protein from the soluble (cytosolic) to the sedimentable (organelle-containing) cell fraction, using leupeptin or other proteinase inhibitors to block inactivation and degradation of the protein inside autophagic vacuoles. A convenient separation method is electrodisruption of the cells, followed by sedimentation of the organelle fraction through a Nycodenz density cushion. A promising variant of the cargo assay is to use a protein probe that is processed by the autophagic-lysosomal pathway so as to generate an intravacuolar fragment. Because there is no cytosolic background, subcellular fractionation is unnecessary, allowing the use of the autophagic fragment assay to measure autophagic activity in whole cells. In hepatocytes, a small fragment, p10(BHMT), made by autophagic processing of the enzyme betaine:homocysteine methyltransferase, thus accumulates in an autophagy-dependent manner in the presence of leupeptin. Autophagic sequestration can also be measured by using exogenous cargo probes, such as radiolabeled di- and trisaccharides, which can be loaded into the cytosol of hepatocytes by reversible electrodisruption or mechanical stress. Raffinose is the preferable probe for measurement of autophagic activity, whereas sucrose (which can be hydrolyzed in amphisomes and lysosomes by added endocytosed invertase) and lactose (which is hydrolyzed in lysosomes by the endogenous beta-galactosidase) are useful for dissection of the various steps in the autophagic-lysosomal pathway and for studying autophagic-endocytic interactions. Furthermore, the intralysosomal hydrolysis of autophagocytosed lactose can be measured in whole cells (as formation of the hydrolysis product, galactose), thus providing a background-free assay (autophagic lactolysis) of the overall autophagic-lysosomal pathway.

Phosphorylated and non-phosphorylated forms of catechol O-methyltransferase in rat liver, brain and other tissues.

Seven different forms of the enzyme COMT (catechol O-methyltransferase) were found in isolated rat hepatocytes by two-dimensional gel electrophoresis and immunoblotting: five small variants (S-COMT) and two large variants (L-COMT). The identities of these COMT forms were verified by tryptic fingerprinting using MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, and by amino acid sequencing using ESI-IT-MS/MS (electrospray ionization with ion-trap tandem MS). Analysis of tissue distributions showed that the S-COMT forms were highly expressed in liver and kidney, whereas L-COMT was expressed more strongly in other tissues. Both of the L-COMT forms were found in all of the tissues examined except the heart, which expressed only the most acidic form, and the kidney, which expressed only the most basic form. Subcellular fractionation revealed the presence of both S-COMT and L-COMT in soluble, as well as sedimentable, fractions, suggesting that they should be classified by size rather than (as previously) by localization. Several of the S-COMT forms were N-acetylated, and the two most acidic forms were found to be phosphorylated at Ser(260). One of the latter was unique to liver cells; the other was also found in kidney, brain and thymus. Among the non-phosphorylated S-COMT forms, one was ubiquitous, one was found in testis and liver, and a third was found in liver, kidney and thymus. No other phosphorylated sites were found, suggesting that the pI differences distinguishing between the various COMT forms are due to some as yet unidentified structural modification(s).

Proteomic analysis of membrane-associated proteins from rat liver autophagosomes.

Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pl 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function. Eleven proteins were highly enriched (consistent with the theoretical maximum of 25x), corresponding to an exclusive membrane localization in the delimiting membrane of the autophagosome. Three of these were methyltransferases: betaine:homocysteine methyltransferase (five variants); catechol O-methyltransferase (one phosphorylated and one unphosphorylated variant) and methionine adenosyltransferase, perhaps indicating that methylation/demethylation of membrane components could play a role in autophagy. A fourth highly enriched autophagosomal protein, phosphatidylethanolamine binding protein, is particularly interesting considering that the autophagic marker protein, LC3/ Atg8, is linked to autophagosomal membranes through its covalent conjugation with phosphatidylethanolamine (as the form LC3-II). LC3-II was not detectable on silver-stained 2D-gels, but could be shown by immunoblotting to be highly enriched in autophagosomal membranes. Other highly enriched proteins were heat shock cognate protein Hsc70 (one short and one long variant), peroxiredoxin 2, peroxiredoxin 6 (two variants), fructose 1,6-bisphosphatase (one phosphorylated and one unphosphorylated variant), adenosine kinase, inorganic pyrophosphatase and selenium-binding protein 2. Hsc70, a chaperonin that plays an important role in the recognition and proteasomal degradation of aggregated proteins as well as in the lysosomal membrane uptake and degradation of certain cytosolic proteins (chaperone-mediated autophagy), could conceivably also serve a recognition function in the autophagic scavenging of denatured or aggregated proteins (aggrephagy). The moderately enriched (2-14x) autophagosomal membrane-associated proteins included a remarkably high proportion of drug-metabolizing enzymes, such as several glutathione S-transferases, sulfotransferases and aromatic hydrocarbon/steroid oxidoreductases. If the autophagic function of these proteins is to recognize protein-drug adducts, they may, along with the peroxiredoxins, chaperonins and methyl metabolic enzymes, make the phagophores (the sequestering precursors of the autophagosomal delimiting membrane) well equipped for the detection and scavenging of proteins denatured by oxidation, hypermethylation, drug adduction or other mechanisms.

Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels.

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.