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1.
Figure 3

Figure 3. From: Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

Nanoliposomal-C6 ceramide delivery resulted in the preferential accumulation of C6 in the mitochondria in leukemic NK cells. Confocal microscopic image of 25μM nanoliposomal-NBD-C6 delivery to RNK16, a rat NK LGL leukemia cell line, showing that NBD-C6 (green) colocalized with cellular mitochondria (red); Cellular nucleus was stained with 4′,6-diamidino-2-phenylindole (blue). Magnification, 63×.

Xin Liu, et al. Blood. 2010 November 18;116(20):4192-4201.
2.
Figure 2

Figure 2. From: Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

Nanoliposomal-C6 ceramide inhibits the activation of ERK, which subsequently down-regulates survivin expression. (A) Western blot analysis was performed for p-ERK after treatment of RNK-16 cells with 5μM ghost or C6 nanoliposome for 18 hours. The equal loading of protein was confirmed by β-actin probing. (B) Western blot analysis was performed for p-ERK, total ERK, or survivin after treatment of NKL cells with different doses of PD098059 for 18 hours. The equal loading of protein was confirmed by β-actin probing. (C) Western blot analysis was performed for survivin after treatment of NKL cells with 25μM ghost (G) or C6 nanoliposome (C6) for 6, 16, and 24 hours. The equal loading of protein was confirmed by β-actin probing. (D) Western blot analysis was performed for survivin after treatment of RNK-16 cells with different doses of either ghost or C6 nanoliposome for 24 hours. The equal loading of protein was confirmed by β-actin probing. (E) Quantitative real-time RT-PCR was performed to measure levels of survivin mRNA after treatment of NKL cells with 25μM ghost or C6 nanoliposome for 6 and 16 hours. *P < .05 indicates significance between ghost and C6 nanoliposome treated samples (Student t test). (F) Quantitative real-time RT-PCR was performed to measure levels of survivin mRNA after treatment of RNK-16 cells with 5 and 20μM ghost or C6 nanoliposome for 24 hours. *P < .05 indicates significance between ghost and C6 nanoliposome-treated samples (Student t test).

Xin Liu, et al. Blood. 2010 November 18;116(20):4192-4201.
3.
Figure 5

Figure 5. From: Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

Nanoliposomal C6-ceramide liposome treatment induces complete remission in LGL leukemic rats. (A). The Kaplan-Meier survival curves for normal rats (n = 14) or leukemic rats without treatment (n = 14) or with treatment of 40 mg/kg ghost (n = 14) or C6 nanoliposome (n = 14), were plotted. Dashed arrow indicates the start of the treatment. (B) Maintenance of normal white blood counts, hemoglobin values, and platelet counts in responding leukemic rats treated with nanoliposomal C6-ceramide. Blood (200μL) from untreated leukemic rats (n = 14), leukemic rats treated with ghost nanoliposome (n = 14), C6-ceramide nanoliposome (n = 14), and normal rats (n = 14) was collected every week from tail veins of the animals and placed in EDTA (ethylenediaminetetraacetic acid) K2–coated tubes, then complete blood count analysis was performed. Arrow indicates the cessation of the treatment. (C) In vivo therapy with nanoliposomal C6-ceramide leads to resolution of organomegaly in responding LGL leukemic rats. The weight of spleen, liver, and thymus were measured in untreated leukemic rats (n = 14), leukemic rats treated with ghost nanoliposome (n = 14), C6-ceramide nanoliposome-responsive rats (n = 5), and normal rats (n = 14). *P < .05, ***P < .0005 indicates significance between ghost and C6-ceramide nanoliposome–treated samples (unpaired t test). D). Flow cytometry was used to identify rat LGL leukemic cells, which are CD3CD8a+. Comparison of CD3CD8a+ NK cells isolated from multiple tissues among normal rats (n = 14), leukemic rats treated with ghost (n = 14), or from rats responsive to C6 nanoliposome treatment (n = 5). Note: elimination of leukemic cells after nanoliposomal C6-ceramide therapy in PBMCs, marrow, and lung. Values shown are the mean, with SD in parentheses. ***P < .0005 indicates significance between ghost and C6-ceramide nanoliposome–treated samples (unpaired t test).

Xin Liu, et al. Blood. 2010 November 18;116(20):4192-4201.
4.
Figure 6

Figure 6. From: Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

Elimination of leukemic infiltration associated with decreased survivin expression and induction of apoptosis in spleens of leukemic rats responsive to nanoliposomal C6 ceramide. Images were taken from hematoxylin and eosin or immunohistochemically stained 5-μm paraffin sections of rat spleen tissues and analyzed with Olympus 1 × 51 Microscope (Olympus America) attached to Spot Insight Camera (Diagnostic Instruments). The software for imaging and analyses was SPOT Advanced Plus Imaging software. (A) Spleen sections from rats treated with ghost nanoliposome (Ai); with C6 ceramide nanoliposome, responsive (Aii), or nonresponsive (Aiii); and from normal control rat (Aiv) were stained with hematoxylin and eosin. (B) Spleen sections from rats treated with ghost nanoliposome (Bi); from C6-ceramide nanoliposome responsive (Bii), or nonresponsive (Biii); and from normal control rat (Biv) were stained with anti-survivin antibody (D-8; Santa Cruz Biotechnology). (C) Spleen sections were stained with an In Situ Cell Death Detection kit to assess the degree of cellular apoptosis via TUNEL-TMR red staining (red); 4′,6-diamidino-2-phenylindole-stained nuclei (blue). Spleen sections from rats responsive to nanoliposomal-C6 (Cii) showed more positive TUNEL staining than sections obtained from rats treated with ghost nanoliposome (Ci), from rats not responding to C6-ceramide nanoliposome (Ciii) and from normal control rat (Civ).

Xin Liu, et al. Blood. 2010 November 18;116(20):4192-4201.
5.
Figure 4

Figure 4. From: Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

Nanoliposomal C6 ceramide induces caspase-dependent apoptosis in leukemic NK cells but not normal PBMCs. (A) The PBMC from NK-LGL leukemia patients (CD3CD56+ cells > 80%; patients #1 and #2 with aggressive NK-LGL leukemia, patients #3 and #4 with chronic NK-LGL leukemia) or 2 normal donors #1 and #2 were treated with 12.5, 25, and 50μM ghost or C6-ceramide nanoliposome or dihydro-C6-ceramide or ghost nanoliposome (patients #5 and #6 with chronic NK-LGL leukemia), respectively for 18 hours; then cells were assayed for apoptosis by flow cytometry. *P < .05, **P < .005 indicate significant differences of C6-treated cells versus ghost nanoliposome–treated cells (Student t test). (B) The PBMCs from individual NK-LGL leukemia patients (CD3CD56+ cells > 80%; patients #1 and #2 with aggressive NK-LGL leukemia, and patients #7 and #8 with chronic NK-LGL leukemia) or 2 normal donors #3 and #4 were treated with 25μM C6-ceramide or ghost nanoliposome for 2, 6, and 24 hours; then cells were assayed for apoptosis by flow cytometry. *P < .05, ***P < .0005 indicate significant difference of ghost nanoliposome–treated cells (Student t test). (C) Western blot analysis was performed for caspase-3 after treatment of PBMCs from patient #8, which were treated with 25μM C6-ceramide or ghost nanoliposome for 2, 6, and 24 hours. Data are representative of 4 independent experiments on PBMCs from 4 NK-LGL leukemia patients. (D) NKL cells were exposed to 25μM C6-ceramide nanoliposome, in the absence or presence of various concentrations of z-VAD-fmk, a pan caspase inhibitor. z-VAD-fmk was added 2 hours before ceramide treatment at the indicated concentration. Cell survival was determined 18 hours later by the MTT assay. *P < .05 indicates significant differences of each dose of z-VAD-fmk–treated cells compared with z-VAD-fmk–untreated cells, respectively (Student t test).

Xin Liu, et al. Blood. 2010 November 18;116(20):4192-4201.
6.
Figure 1

Figure 1. From: Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

Role of survivin in leukemic NK cell survival. (A) Quantitative real-time RT-PCR was performed to measure levels of survivin mRNA in PBMCs from NK-LGL leukemia patients (CD3CD56+ > 80%) or purified NK cells isolated from normal donors. Each circle represents an individual purified NK sample. cDNA samples were diluted 1:100 for 18S expression. ***P < .0005 indicates leukemic NK cells versus normal NK cells (Mann-Whitney test). (B) Suspension splenocytes were isolated from spleens of NK-LGL leukemic F344 rats or age- and gender-matched normal rats. RNAs were harvested from these cells, and quantitative real-time RT-PCR was performed to measure levels of survivin mRNA. cDNA samples were diluted 1:100 for 18S expression. **P < .005 indicates leukemic NK cells versus normal NK cells (Mann-Whitney test). (C) Mitochondria were isolated from human aggressive NK-LGL leukemia cell line NKL or rat aggressive NK-LGL leukemia cell line RNK-16, or pooled enriched NK cells (CD3CD56+ 80%-95%) from 5 normal human donors, or 4 individual patients with chronic NK-LGL leukemia (CD3CD56+ cells > 80%), then resolved in the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel loading buffer in a boiling water bath for 5 minutes. Western blot analysis was performed for detection of survivin. Cox IV, a mitochondria marker, was used as loading control. (D) NKL cells were transfected with 10 μg control siRNA or human survivin ON-TARGET plus SMARTpool siRNA (Dharmacon) per 5 × 105 cells, each complexed within cationic nanoliposomes, then MTT assay was performed 72 hours after transfection. **P < .005 indicate significant difference in cell viability of survivin siRNA-transfected cells compared with control siRNA-transfected cells (Student t test). Inset, Western blot analysis was performed for survivin in the control siRNA or survivin siRNA-transfected NKL cells 72 hours after transfection. The equal loading of protein was confirmed by probing with glyceraldehyde 3-phosphate dehydrogenase.

Xin Liu, et al. Blood. 2010 November 18;116(20):4192-4201.

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