Temozolomidsensitivität von Tumorstammzellen im humanen Glioblastom

Eine kleine Gruppe von Tumorstammzellen ist in Glioblastomen für die Aufrechterhaltung des Tumorwachstums verantwortlich. Eine häufig formulierte Hypothese lautet, dass sich die Tumorrezidive auf Grund der verminderten Empfindlichkeit von Tumorstammzellen auf etablierte Therapien bilden. Unklar ist bislang jedoch, inwieweit Temozolomid (Temodal®) tatsächlich wirksam gegen diese Subpopulation ist. Hierzu untersuchte ich fünf verschiedene, aus primären Glioblastomen gewonnene Tumorstammzelllinien. Temozolomid hatte auch in hohen Dosen eine nur sehr geringe Toxizität auf die Gesamtheit aller Zellen. Im Gegensatz hierzu zeigte sich eine signifikant stärkere dosis- und zeitabhängige Abnahme der CD133+ Zellpopulation. In gleicher Weise induzierte Temozolomid dosisabhängig den selektiven Zelltod von Zellen mit Expression des Stammzellmarkers Nestin während Zellen mit Expression von Differenzierungsmarkern (β-III Tubulin, GFAP, GalC) vom Temozolomid-induzierten Zelltod geringer betroffen waren. Parallel zur verminderten Expression von Stammzellmarkern verminderte Temozolomid die Anzahl der in vitro zur Sphärenbildung befähigten Zellen, nachgewiesen im sog. minimal dilution assay, einen häufig verwendeten Assay zur Bestimmung des Stammzellanteils innerhalb einer Zellkultur. Interessanter Weise zeigten mittels FACS aufgereinigte CD133± Zellen keinen signifikanten Unterschied in der Temozolomidempfindlichkeit, als ursächlich hierfür stellte sich eine rasche Differenzierung der CD133+ Tumorstammzellen in CD133- Zellen heraus. Zusammenfassend zeigten die bisherigen Daten, dass Tumorstammzellen im Glioblastom entgegen meinen Erwartungen eine höhere Empfindlichkeit gegenüber Temozolomid als Tumorzellen ohne Stammzelleigenschaften besitzen. Dies lässt vermuten, dass durch eine Optimierung der aktuellen Temozolomid Dosierungsschemata die Wirksamkeit der Therapie auf Tumorstammzellen signifikant gesteigert und damit das Überleben der Patienten verbessert werden kann

Differential basolateral–apical distribution of scavenger receptor, class B, type I in cultured cells and the liver

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral–apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro. Electronic supplementary material The online version of this article (doi:10.1007/s00418-014-1251-9) contains supplementary material, which is available to authorized users

Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other

Differential basolateral–apical distribution of scavenger receptor, class B, type I in cultured cells and the liver

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral–apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro.ISSN:0948-6143ISSN:1432-119

GW4064 and CDCA reduce CD36 expression and function.

<p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours and gene expression was analyzed by qRT-PCR (n = 3). (b) Cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hrs and protein expression was determined by western blot analysis and results were quantitated by densitometry (n = 3). (c) Fatty-acid uptake was determined after treatment with 10 µM GW4064 or 100 µM CDCA as described in the methods section (n = 3).</p

Bile acids and a non-steroidal FXR agonist reduce HDL endocytosis.

<p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours. Gene expression was analyzed by qRT-PCR and expression levels were normalized to GAPDH expression (n = 2). The increase in SHP mRNA indicates FXR activation. (b) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 50 µg/ml HDL-Alexa⁴⁸⁸ for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = 10 µm. (c) Quantification of fluorescence intensities of (b). (d) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 µg/ml ¹²⁵I-HDL for 1 hour. Uptake was determined after displacing cell surface bound HDL by a 100-fold excess at 4°C for 1 hour (n = 3).</p

GW4064 and CDCA reduce HDL endocytosis independently of SR-BI.

<p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours and gene expression was analyzed by qRT-PCR (n = 3). (b) Cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hrs and protein expression was determined by western blot analysis and results were quantitated by densitometry (n = 3). HepG2 cells transfected with scrambled shRNA (c) or SR-BI shRNA (d) were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 µg/ml double labeled ¹²⁵I/³H-CE-HDL for 1 hr. Selective cholesteryl-ester uptake was calculated by subtracting ¹²⁵I-HDL uptake from ³H-CE-HDL uptake (n = 3).</p

Modification of HDL by taurocholate does not alter endocytosis.

<p>(a) HDL was incubated with or without 1 mM taurocholate in media in the absence of cells for 1 hour. HDL size was then analyzed by size exclusion chromatography. HDL incubated with taurocholate is eluted earlier, indicating increased size. (b) HDL-Alexa⁴⁸⁸ was incubated with or without 1 mM taurocholate in media in the absence of cells for 1 hour. Free taurocholate was then removed using gel filtration and HepG2 cells were incubated with this modified HDL-Alexa⁴⁸⁸ for 1 hour. Cells were fixed, counterstained with DAPI and imaged. (c) Quantification of fluorescence intensities from (b); n = 3. Green: HDL; blue: nucleus; bar = 10 µm.</p

Taurocholate reduces HDL endocytosis SR-BI-dependently.

<p>(a) HepG2 cells were incubated with or without 1 mM taurocholate and ATP hydrolysis was measured as a decrease in extracellular ATP. One representative experiment out of three independent experiments is shown. (b) SR-BI knockdown efficiency in HepG2 cells transfected with scrambled shRNA and HepG2 cells transfected with SR-BI shRNA (n = 3). Selective lipid uptake analysis using double labeled ¹²⁵I/³H-CE-HDL in scrambled control (c) or SR-BI knockdown (d) HepG2 cells (n = 3). Selective cholesteryl-ester uptake was calculated by subtracting ¹²⁵I-HDL uptake from ³H-CE-HDL uptake.</p

Taurocholate neither exerts cytotoxic effects, nor inhibits transferrin or LDL endocytosis in HepG2 cells.

<p>(a) Cells were incubated with the indicated concentrations of taurocholate for 1 hour. No release of LDH into the cell culture supernatant was detected. 0.1% Triton-X100 was used as a positive control. (b) Cells were incubated with 20 µg/ml transferrin-Alexa⁴⁸⁸ (b) or 50 µg/ml LDL-Alexa⁵⁶⁸ (c) with or without 1 mM taurocholate at 37°C for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: transferrin; red: LDL; blue: nucleus; bar = 10 µm. Neither transferrin nor LDL uptake were altered. Quantifications of fluorescent signals are depicted next to the images. (d) Cells were incubated with or without 1 mM taurocholate for 1 hour. Cells were fixed, stained with Filipin and imaged. Bar = 10 µm. Representative images of 3 independent experiments are shown.</p