Nitric oxide from rat liver sinusoidal endothelial cells induces apoptosis in IFN γ- sensitized CC531s colon carcinoma cells  Katrien Vekemans, Filip Braet, David Muyllaert, Eddie Wisse  Journal of Hepatology  Volume 41, Issue 1, Pages 11-18 (July 2004) DOI: 10.1016/j.jhep.2004.03.026

Fig. 1 RT-PCR and immunocytochemical evaluation of FasL expression on isolated rat LSECs. (A) RT-PCR was performed on mRNA. Reverse-transcription and amplification were performed as described in material and methods with primers specific for FasL. Lane A: 100 bp molecular marker. Lane B: negative control, PCR reaction without RNA template. Lane C: FasL of LSECs mRNA (40 cycles). Lane D: β-actin of LSECs mRNA (internal control, 28 cycles). (B) CLSM revealed positive staining of the LSECs for FasL in the cytoplasm, the nucleus was stained red by propidium iodide. Note that the positivity was visual at one side of the cell. Data were obtained from at least three independent experiments. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 2 Quantification of apoptosis measured as % specific DNA fragmentation. CC531s were cultured in the presence of 1, 10 and 100 ng/ml hrFasL with and without enhancer and with IFNγ (100 U/ml). Apoptosis was induced and a dose-dependent curve was obtained. Note the significant difference (Student's t-test) between 1 and 100 ng/ml (P≤0.03, Student's t-test). **, significantly different from 100 ng with enhancer (P≤0.01, Student t-test); (n=4), mean±standard error. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 3 Effect of IFNγ on the functionality of Fas on CC531s. (A) Condition with IFNγ (100 U/ml), (B) 100 ng/ml hrFasL+enhancer (1 μg/ml)+IFNγ (100 U/ml). HO33342 blue stain; PI red stain. CC531s with fragmented blue (arrowpoint) nuclei are apoptotic and CC531s without a sign of nuclear fragmentation and blue are live cells (asterisk). An enhanced amount of apoptotic cells can be noticed from A to B. (C) Control condition with IFNγ (100 U/ml), (D) 100 ng/ml hrFasL+enhancer (1 μg/ml)+IFNγ (100 U/ml). When hrFasL is added, the CC531s retract and reveal blebs on the cell membrane. Moreover, they detach from the culture dish. This in comparison to the control condition where the CC531s reveal normal hairy surface and flat appearance like the cell on the left hand side. Scale bars, 10 μm. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 4 Electron microscopic evaluation after 18hrs of co-culture of LSECs and IFNγ-pretreated CC531s revealed enhanced apoptosis on CC531s. (A) TEM; CC531s cell revealed fragmented nucleus and vacuolization in the cytoplasm. (B) SEM; CC531s (CC) revealed blebbing and detachment (arrowpoints) from the culture dish, while LSECs remained spread and intact. Note the clearly visible nucleus (N) of the LSECs. Scale bars, 10 μm. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 5 Apoptosis in CC531s after co-culture ‘LSECs: CC531s’ and ‘LSECs: IFNγ-pretreated CC531s’. ‘LSECs: CC531s’: No apoptosis could be quantified, measured by specific DNA fragmentation assay. ‘LSECs: IFNγ-pretreated CC531s’: Apoptosis could be measured in a ratio-dependent curve. When treated with aFasL (20 μg/ml) blocking the Fas/FasL pathway, no decrease in the apoptosis could be measured. However, when the co-cultures ‘LSECs: IFNγ-pretreated CC531s’ where treated with NO inhibitors NMMA (1 mM) and DEX (10 μg/ml), the apoptosis was inhibited. * Significantly different from medium condition in corresponding ratio (P≤0.05, Student's t-test), ** Significantly different from IFN condition in corresponding ratio (P≤0.05, Student's t-test) (n=3), mean±standard error. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 6 Effect of NMMA and DEX on LSECs and CC531s separately. (A) NMMA nor DEX had effect on the CC531s, measured by specific DNA fragmentation assay. (B) For LSECs the same conclusion could be made, measured by an endocytosis assay. All the LSECs remained attached to the culture dish. (n=3). The measurement resulted in 106±11% up-take of FluoSpheres when the control condition is considered 100% up-take. Mean±standard error. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 7 Bar graph showing the production of NO in co-cultures of CC531s and LSECs; and in mono-cultures of LSECs, CC531s and IFNγ-pretreated CC531s (n=3). NO was present in the medium in co-cultures of LSECs and IFNγ-pretreated CC531s. In co-cultures of LSECs and in IFNγ-pretreated CC531s, NO was also present in the medium in an amount not significantly different from above. Control CC531s produced NO and not significantly different when they were pretreated with IFNγ. Inset: mono-cultures of LSECs show production of NO (control). The amount of LSECs was identical as used in the ratio-depending co-cultures. (n=3), mean±standard error. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 8 In vivo: CC531s were injected in the mesenteric vein and after 17 days the metastasis was evaluated. IFNγ-pretreated CC531s (A) were injected and metastasis still occurred, but was less evaluated compared to B, where untreated CC531s were injected. Also liver weight was significantly different in both conditions (P≤0.0006, Student's t-test), (n=4), mean±standard error. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)

Fig. 9 Schematic overview of the liver sinusoid. LSECs express FasL and CC531s express Fas. When IFNγ is present in the sinusoid, Fas becomes active. NO produced by the LSECs induces apoptosis in CC531s cells only when IFNγ is present. By this means the IFNγ-activated pathway supports the immune system by killing tumor cells. PC: parenchymal cell. Journal of Hepatology 2004 41, 11-18DOI: (10.1016/j.jhep.2004.03.026)