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Diffusion of inner membrane proteins is a prerequisite for correct functionality of mitochondria. The complicated structure of tubular, vesicular or flat cristae and their small connections to the inner boundary membrane impose constraints on the mobility of proteins making their diffusion a very complicated process. Therefore we investigate the molecular transport along the main mitochondrial axis using highly accurate computational methods. Diffusion is modeled on a curvilinear surface reproducing the shape of mitochondrial inner membrane (IM). Monte Carlo simulations are carried out for topologies resembling both tubular and lamellar cristae, for a range of physiologically viable crista sizes and densities. Geometrical confinement induces up to several-fold reduction in apparent mobility. IM surface curvature per se generates transient anomalous diffusion (TAD), while finite and stable values of projected diffusion coefficients are recovered in a quasi-normal regime for short- and long-time limits. In both these cases, a simple area-scaling law is found sufficient to explain limiting diffusion coefficients for permeable cristae junctions, while asymmetric reduction of the junction permeability leads to strong but predictable variations in molecular motion rate. A geometry-based model is given as an illustration for the time-dependence of diffusivity when IM has tubular topology. Implications for experimental observations of diffusion along mitochondria using methods of optical microscopy are drawn out: a non-homogenous power law is proposed as a suitable approach to TAD. The data demonstrate that if not taken into account appropriately, geometrical effects lead to significant misinterpretation of molecular mobility measurements in cellular curvilinear membranes.
Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component ({tau}1) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions {tau}2 and {tau}3 increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of {pi}-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.
High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.
Elevated tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. Clinically, TIFP may hamper the uptake of chemotherapeutic drugs into the tumor tissue reducing their therapeutic efficacy. In this study, a means of modulating TIFP to increase the flux of macromolecules into tumor tissue is presented, which is based on the rationale that elevated plasma colloid osmotic pressure (COP) pulls water from tumor interstitium lowering the TIFP. Concentrated human serum albumin: (20% HSA), used as an agent to enhance COP, reduced the TIFP time-dependently from 8 to 2 mm Hg in human tumor xenograft models bearing A431 epidermoid vulva carcinomas. To evaluate whether this reduction facilitates the uptake of macromolecules, the intratumoral distribution of fluorescently conjugated dextrans (2.5 mg/ml) and cetuximab (2.0 mg/ml) was probed using novel time domain nearinfrared fluorescence imaging. This method permitted discrimination and semiquantification of tumor-accumulated conjugate from background and unspecific probe fluorescence. The coadministration of 20% HSA together with either dextrans or cetuximab was found to lower the TIFP significantly and increase the concentration of the substances within the tumor tissue in comparison to control tumors. Furthermore, combined administration of 20%HSA plus cetuximab reduced the tumor growth significantly in comparison to standard cetuximab treatment. These data demonstrate that increased COP lowers the TIFP within hours and increases the uptake of therapeutic macromolecules into the tumor interstitium leading to reduced tumor growth. This model represents a novel approach to facilitate the delivery of therapeutics into tumor tissue, particularly monoclonal antibodies.
Elevated tumor interstitial fluid pressure (TIFP) is a prominent feature of solid tumors and hampers the transmigration of therapeutic macromolecules, for example, large monoclonal antibodies, from tumor-supplying vessels into the tumor interstitium. TIFP values of up to 40 mm Hg have been measured in experimental solid tumors using two conventional invasive techniques: the wick-in-needle and the micropuncture technique. We propose a novel noninvasive method of determining TIFP via ultrasonic investigation with scanning acoustic microscopy at 30-MHz frequency. In our experimental setup, we observed for the impedance fluctuations in the outer tumor hull of A431-vulva carcinoma–derived tumor xenograft mice. The gain dependence of signal strength was quantified, and the relaxation of tissue was calibrated with simultaneous hydrostatic pressure measurements. Signal patterns from the acoustical images were translated into TIFP curves, and a putative saturation effect was found for tumor pressures larger than 3 mm Hg. This is the first noninvasive approach to determine TIFP values in tumors. This technique can provide a potentially promising noninvasive assessment of TIFP and, therefore, can be used to determine the TIFP before treatment approach as well to measure therapeutic efficacy highlighted by lowered TFP values.
The mechanistic target of rapamycin (mTOR) is elevated in prostate cancer, making this protein attractive for tumor treatment. Unfortunately, resistance towards mTOR inhibitors develops and the tumor becomes reactivated. We determined whether epigenetic modulation by the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), may counteract non-responsiveness to the mTOR inhibitor, temsirolimus, in prostate cancer (PCa) cells. Prostate cancer cells, sensitive (parental) and resistant to temsirolimus, were exposed to VPA, and tumor cell growth behavior compared. Temsirolimus resistance enhanced the number of tumor cells in the G2/M-phase, correlating with elevated cell proliferation and clonal growth. The cell cycling proteins cdk1 and cyclin B, along with Akt-mTOR signaling increased, whereas p19, p21 and p27 decreased, compared to the parental cells. VPA significantly reduced cell growth and up-regulated the acetylated histones H3 and H4. Cdk1 and cyclin B decreased, as did phosphorylated mTOR and the mTOR sub-complex Raptor. The mTOR sub-member Rictor and phosphorylated Akt increased under VPA. Knockdown of cdk1, cyclin B, or Raptor led to significant cell growth reduction. HDAC inhibition through VPA counteracts temsirolimus resistance, probably by down-regulating cdk1, cyclin B and Raptor. Enhanced Rictor and Akt, however, may represent an undesired feedback loop, which should be considered when designing future therapeutic regimens.
CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins
(2006)
The mechanisms leading to prostate cancer metastasis are not understood completely. Although there is evidence that the CXC chemokine receptor (CXCR) 4 and its ligand CXCL12 may regulate tumor dissemination, their role in prostate cancer is controversial. We examined CXCR4 expression and functionality, and explored CXCL12-triggered adhesion of prostate tumor cells to human endothelium or to extracellular matrix proteins laminin, collagen, and fibronectin. Although little CXCR4 was expressed on LNCaP and DU-145 prostate tumor cells, CXCR4 was still active, enabling the cells to migrate toward a CXCL12 gradient. CXCL12 induced elevated adhesion to the endothelial cell monolayer and to immobilized fibronectin, laminin, and collagen. Anti-CXCR4 antibodies or CXCR4 knock out significantly impaired CXCL12-triggered tumor cell binding. The effects observed did not depend on CXCR4 surface expression level. Rather, CXCR4-mediated adhesion was established by alpha5 and beta3 integrin subunits and took place in the presence of reduced p38 and p38 phosphorylation. These data show that chemoattractive mechanisms are involved in adhesion processes of prostate cancer cells, and that binding of CXCL12 to its receptor leads to enhanced expression of alpha5 and beta3 integrins. The findings provide a link between chemokine receptor expression and integrin-triggered tumor dissemination.
The genome, antigens of human cytomegalovirus (HCMV) are frequently found in prostatic carcinoma. However, whether this infection is causative or is an epiphenomenon is not clear. We therefore investigated the ability of HCMV to promote metastatic processes, defined by tumor cell adhesion to the endothelium, extracellular matrix proteins. Experiments were based on the human prostate tumor cell line PC3, either infected with the HCMV strain Hi (HCMVHi) or transfected with cDNA encoding the HCMV-specific immediate early protein IEA1 (UL123) or IEA2 (UL122). HCMVHi upregulated PC3 adhesion to the endothelium, to the extracellular matrix proteins collagen, laminin, fibronectin. The process was accompanied by enhancement of β1-integrin surface expression, elevated levels of integrin-linked kinase, phosphorylation of focal adhesion kinase. IEA1 or IEA2 did not modulate PC3 adhesion or β1-integrin expression. Based on this in vitro model, we postulate a direct association between HCMV infection, prostate tumor transmigration, which is not dependent on IEA proteins. Integrin overexpression, combined with the modulation of integrin-dependent signalling, seems to be, at least in part, responsible for a more invasive PC3Hi tumor cell phenotype. Elevated levels of c-myc found in IEA1-transfected or IEA2-transfected PC3 cell populations might promote further carcinogenic processes through accelerated cell proliferation.
Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells
(2010)
Background: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. Methodology/Principal Findings: The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. Conclusion/Significance: Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.
Background: The treatment of different skin conditions with spa waters is a long tradition dating back to at least late Hellenism. Interestingly, independent scientific examinations studying the effect of spa waters are scarce.
Objective: In the present in vitro study, we compared the effect of culture media supplemented with (a) thermal spa waters (La Roche-Posay, Avène) and (b) two natural mineral drinking waters (Heppinger, Adelholzener) on physiological parameters in HaCaT keratinocytes.
Methods: The different medium preparations were investigated with regard to cell proliferation and cell damage. Moreover, the impact on inflammation parameters with and without ultraviolet B (UVB) irradiation was examined.
Results: Two popular thermal spring waters were found to suppress cell proliferation and cell damage. Moreover, these waters reversed the induction of interleukin-6, as measured using enzyme-linked immunosorbent assay and promoter transactivation, and the formation of reactive oxygen species after UVB stimulation. Of note, the two natural mineral waters, which are distributed as drinking waters, had some effect on the above-mentioned parameters but to a lesser extent.
Conclusion: In summary, our results show that spa waters, and particularly those derived from thermal springs, reduce parameters associated with inflammation. It seems likely that trace elements such as selenium and zinc are critical for the observed effects.