| 1. |
- Dubois, Louise, et al.
(författare)
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Malignant Cell-Derived Extracellular Vesicles Express Different Chromogranin Epitopes Compared to Prostasomes
- 2015
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Ingår i: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 75:10, s. 1063-1073
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND. Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. METHODS. We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. RESULTS. A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. CONCLUSION. Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers.
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| 2. |
- Kharaziha, Pedram, et al.
(författare)
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Sorafenib Has Potent Antitumor Activity against Multiple Myeloma In Vitro, Ex Vivo, and In Vivo in the 5T33MM Mouse Model
- 2012
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 72:20, s. 5348-5362
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Tidskriftsartikel (refereegranskat)abstract
- Multiple myeloma (MM) is a B-cell malignancy characterized by the expansion of clonal plasma blasts/plasma cells within the bone marrow that relies on multiple signaling cascades, including tyrosine kinase activated pathways, to proliferate and evade cell death. Despite emerging new treatment strategies, multiple myeloma remains at present incurable. Thus, novel approaches targeting several signaling cascades by using the multi-tyrosine kinase inhibitor (TKI), sorafenib, seem a promising treatment approach for multiple myeloma. Here, we show that sorafenib induces cell death in multiple myeloma cell lines and in CD138(+)-enriched primary multiple myeloma patient samples in a caspase-dependent and -independent manner. Furthermore, sorafenib has a strong antitumoral and -angiogenic activity in the 5T33MM mouse model leading to increased overall survival. Multiple myeloma cells undergo autophagy in response to sorafenib, and inhibition of this cytoprotective pathway potentiated the efficacy of this TKI. Mcl-1, a survival factor in multiple myeloma, is downregulated at the protein level by sorafenib allowing for the execution of cell death, as ectopic overexpression of this protein protects multiple myeloma cells. Concomitant targeting of Mcl-1 by sorafenib and of Bcl-2/Bcl-xL by the antagonist ABT737 improves the efficacy of sorafenib in multiple myeloma cell lines and CD138(+)-enriched primary cells in the presence of bone marrow stromal cells. Altogether, our data support the use of sorafenib as a novel therapeutic modality against human multiple myeloma, and its efficacy may be potentiated in combination with ABT737.
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| 3. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 4. |
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| 5. |
- Panaretakis, Theocharis
(författare)
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On the pro-apoptotic mechanisms of the antitumor drugs doxorubicin and interferon-alpha
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anti-cancer drugs act primarily by inducing apoptosis. However, knowledge of how various substances induce apoptosis is still incomplete, and so is the basis for the great variation in cellular sensitivity to cytotoxic drugs. A detailed understanding of how anti-cancer agents induce cell death and how defects in cell death pathways promote resistance will change the way chemotherapeutic drugs are used and designed. The aim of this thesis was to investigate pro-apoptotic signaling induced by two commonly used anti-cancer drugs, Doxorubicin and Interferon-alpha. Doxorubicin (DXR), an anthracycline, is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. Interferon-alpha (IFN-alpha) is a pleiotropic cytokine and its ability to induce apoptosis has been proposed to be of major importance for its clinical anti-tumor activity. The results demonstrate that the mechanisms of induction of apoptosis by both drugs are strikingly similar with respect to the signaling involved. Clinically relevant concentrations of both agents induce the activation of the pro-apoptotic Bcl-2 family members Bak and Bax prior to apoptosis and anti-apoptotic Bcl-2 family members regulate this response. We could also demonstrate that Bak is activated prior to Bax by both agents. Upstream of Bak, Bax and the mitochondria, two kinases that are known to be activated by cellular stress, JNK and PKCdelta, are involved, both with respect to DXR and IFN-alpha. We demonstrated the requirement of Bak and Bax for the induction of apoptosis by DXR by using bax- as well as bak-deficient mouse embryo fibroblasts (MEFs). The BH3-only protein, Bik, which is induced in response to DXR, could be an activator of Bak and Bax. Upstream of the Bcl-2 family members, caspase-2 is activated and was found to be required for DXR-induced apoptosis in Jurkat cells. PKCdelta was found to be one of the critical downstream targets of caspase-2 following DXR treatment. By using chemical inhibitors against caspase-2, PKCdelta and JNK, our data suggest a signaling model involving caspase-2, PKCdelta and JNK. Survival signaling could mask the true potential of chemotherapeutic agents as demonstrated by co-incubation of a PI3K-inhibitor with DXR. Inhibition of PI3K potentiated the DXR-induced Bak, Bax activation and apoptosis in a Bcl-2 dependent but in a caspase-2, JNK and PKCdelta-independent manner. The upstream signaling in IFN-alpha-induced apoptosis was also addressed. Upstream of the mitochondria, IFN-a induces JNK phosphorylation/activation. Inhibition of JNK significantly blocked IFN-alpha-induced Bak and Bax activation and apoptosis, but did not affect the IFN-alpha-stimulated Jak/STAT signaling. This suggests that the canonical IFN-alpha induced pathway is not sufficient for this response. Inhibition of JNK was also found to influence the phosphorylation of the pro-apoptotic PKC family member, PKCdelta. Furthermore, PKCdelta inhibition blocked apoptosis and Bak activation induced by IFN-alpha. We conclude that IFN-alpha-induced apoptosis involves the mitochondrial pathway and the kinases JNK and PKCdelta. Furthermore this stress-related IFN-induced pathway is unrelated to the Jak/STAT signaling which is generally thought to mediate IFN-alpha's cellular responses.
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| 6. |
- Ronquist, Karl Göran, et al.
(författare)
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Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells
- 2016
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process.
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| 7. |
- Söderberg, Lovisa, 1988-, et al.
(författare)
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Detection of single exosomes in microfluidic droplets by RT-PCR amplification of 18S RNA content
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We present a workflow for reverse transcription-PCR (RT-PCR) in microfluidic droplets to identify exosomes based on their RNA content. Available techniques for exosome detection have been limited to size or surface markers which limit their diagnostic capabilities. Exosome detection based on RNA content could be developed to be used as a diagnostic, prognostic or predictive tool for cancer based on specific RNA biomarkers in liquid biopsies. In this manuscript we demonstrate a high throughput method for the amplification of exosome derived 18S RNA in microfluidic droplets. Automated image analysis using open source software was applied to distinguish and count PCR-positive droplets with fluorescent intensity over a set threshold. We benchmark our workflow against picoliter scale RT-PCR on serially diluted exosome samples and demonstrate the ability of the droplet based workflow to correctly rank exosome samples based on exosome concentration. This represents a key step towards a quantitative analysis of exosomal RNA content and the sorting of single exosomes by their RNA content.
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