ATIII is predominately in its S-configuration in blood at a physiological concentration of about 2.4 mM. To determine whether modification of ATIII has an effect on in vivo therapeutic HIV activity, we assessed three forms of the protein: non-activated ATIII, heparin-activated ATIII – both given intravenously – and liposomally encapsulated ATIII given subcutaneously. Nonactivated ATIII, at a concentration sufficient to reduce inflammation in a baboon model of sepsis [34], and at 10?0-fold normal physiologic concentrations, did not impede viral replication. In vitro experiments had demonstrated that the anti-HIV activity of ATIII could be enhanced through heparin activation [16], and in concordance with this we found that intravenous administration of hep-ATIII resulted in a modest inhibition of viral replication in vivo, confirming the importance of ATIII activation. However, we observed the most potent inhibition of plasma virus when ATIII was packaged in immunoliposomes and delivered subcutaneously. There are several possible explanations for this observation: (1) It is likely that unencapsulated hep-ATIII is not specifically transported to lymph nodes, a tissue that harbors viral replication [65], while in contrast, anti-HLA-DR immunoliposomes likely transport ET-ATIII to this location [19]. (2) Follicular dendritic cells (FDC) may stimulate viral replication in lymphocytes, and it has been demonstrated that serpins may interfere with this process [66].
Detection of caspase signaling by western blot analysis 24 hours after different concentrations of LUT (10?00 mM). UVB (120 mJ/cm2) was used as a positive control for cell death in NHK. D. Luminescent detection of caspase 8 and caspase 9 activity 24 hours after treatment with 50 mM LUT. Fold increase values against untreated control are shown. (Experiment performed three times in duplicate) E. Measurement of viability by trypan blue exclusion assay, 24 hours after treatment with LUT (50 mM) and zVAD-fmk (50 mM). LUT treatment increased the lysosomal compartment in MET4 cells
Besides the typical morphological changes accompanying the apoptotic phenotype induced by LUT, we also observed in the cytoplasm of the MET4 cells the formation of vacuoles (Figure 3Afirst row). Staining the LUT-treated MET4 cells with acridine orange (AO), identified those vesicles as acidic vesicular organelles (AVO) (Figure 3A, AVO’s in red), suggesting an increase of the lysosomal compartment. Quantification of AVO’s by flow cytometric analysis of LUT-treated MET1, MET4 and NHK-
cells showed a dose-dependent increase of AVO’s which was more pronounced in MET4 cells. In contrast, we could not detect an increase of AVO’s in NHKs (Figure 3B). Lysosomes are crucially important for catabolic processes such as autophagy, which is a survival mechanism involved in the breakdown and recycling of damaged or potentially dangerous proteins and organelles in response to stress [22,28]. Moreover, it was recently discovered that an increase in the number of perinuclear lysosomes makes cells more prone for autophagic flux, as fusion between autophagosomes and lysosomes becomes more likely [29,30].
Figure 2. LUT induced apoptosis involves modulation of AKT signaling. A. Time dependent effect of LUT on AKT phosphorylation (AKT-P; Ser473) and on apoptosis markers (Parp-cleavage and caspase 3 activation) was studied using western blot in MET1 and MET4 cells B. Protein and phosphorylation levels of AKT and its downstream targets in MET1 and MET4 cells treated with LUT (20 and 50 mM) for 24 hours and 48 h obtained using western blot analysis. Numbers, obtained by densitometric analysis of the western blot, indicate the ratio of phosphorylated to total AKT and p70S6 protein. C. MET1 and MET4 cells were pre-treated or not with AI (10 mM) for 1 hour and subsequently treated with LUT as indicated. Densitometric analysis of cleaved caspase 3 relative to actin level is shown. A representative blot of at least 3 independent experiments is shown. D. Cells were treated with AI and LUT for 24 hours as indicated and the amount of apoptotic DNA was determined by the Cell death detection ELISA as described in Materials and Methods. Experiment was performed twice in duplicate E. MET1 and MET4 cells transiently transfected with a construct expressing Myr-AKT-HA, were treated or not with LUT (50 mM) for 24 hours. Fugene HD = transfectant only; Myr-AKT = constitutively active AKT). Densitometric analysis of cleaved caspase 3 relative to actin level is shown. A representative blot of at least 3 independent experiments is shown. treated cells, we stained lysosomes using Lysosome Associated Membrane Protein (LAMP)-1 and LAMP-2 directed antibody and used DAPI for nuclear staining. These stainings revealed an increase in number and in size of lysosomes around the nucleus of MET4 cells following LUT-treatment (Figure 3C).Hence, the observed massive increase of the lysosomal compartment in MET4 as a result of LUT treatment, suggests the stimulation of a catabolic process dependent on lysosomal degradation.
Figure 3. Expanded lysosomal compartment following LUT-treatment in metastatic SCC cells. A. First row: Bright field microscopic pictures of MET4 treated or not with LUT (50 mM) for 24 hours. Second and third row: Fluorescent microscopic images of AO stained MET4 cells treated with LUT (50 mM) or left untreated. Red = AVO; Green: non-acidic cell compartment (scale bar = 20 mm). B. Representive flow cytometric quantification of AVO formation (increase in red fluorescence in MET1, MET4 and NHKs following treatment with indicated concentrations of LUT C. Confocal images of LAMP-1 and LAMP-2 stained (green) MET4 cells treated with LUT (50 mM) for 16 h. DAPI (blue) was used for nuclear staining.Autophagy is involved in the response of SCC cells to LUT
Reduced AKT/mTOR signaling paralleled by an increase in lysosomal activity points towards the activation of a specific catabolic process, called autophagy, which is known to be a survival mechanism frequently utilized by cancer cells to adapt to metabolic and cellular stress [31,32]. An important hallmark of autophagy is the formation of double membrane vacuoles, called autophagosomes. Ultrastructural analysis of LUT-treated MET4 cells using transmission electron microscopy (TEM) showed increased presence of autophagosomes filled with cargo (Figure 4A). An increase in autophagosome accumulation may result either from increased autophagosome formation or from the blockage of autophagic degradation after fusion of the autophagosome with a lysosome (autolysosome) [33]. The tandem-tagged LC3 construct (tfLC3), mRFP-GFP-LC3, is used to distinguish early and late autophagosomes. The GFP-tag will be quenched quickly in the acidic environment of the autolysosome, leaving only the mRFP-tag detectable [34]. Figure 4B clearly shows the accumulation of mRFP punctae in the absence of green fluorescence in LUTtreated MET4 cells (Figure 4B), indicating an increased autolysosome formation and enhanced autophagic flux. Autophagic flux can also be evaluated by decreased p62 protein levels, since p62 acts as an autophagosomal cargo receptor for ubiquitinated proteins, which is degraded in the autolysosome [35]. Indeed, addition of LUT to MET4 cells decreased p62 levels, albeit only at higher LUT concentrations, suggesting that LUT increases the autophagic flux (Figure 4C). Levels of LC3-II, which is incorporated in the outer and inner membrane of the autophagosomes [36], decreased as well upon treatment with 50 mM of LUT which is possibly due to the massive formation of autolysosomes and the simultaneous induction of apoptosis. In agreement, blocking lysosomal degradation using chloroquine (CQ) rescued LC3-II and p62 breakdown.