MBO-asGCS suppresses tumor growth in athymic nude mice.

<p>Athymic nude mice (<i>Foxn1^nu/Foxn1⁺</i>, 10 per group) bearing drug-resistant tumors (NCI/ADR-RE cells) were treated with either MBOs (1 mg/kg every 3 days, intratumoral injection) alone or MBOs combined with doxorubicin (Dox, 2 mg/kg once a week, intraperitoneal injection) for 38 days. Treatments were started once tumors were visible (2 mm diameter, day 0). a. Tumor growth. *, p<0.001 compared with treatments in the presence of saline or MBO-SC. **, p<0.001 compared with treatments in the presence of doxorubicin or doxorubicin combined with MBO-SC. b. Body weight of mice after treatments. c. Tumors after treatments. Tumors were photographed when mice were sacrificed at the end of treatment regimens.</p

MBO-asGCS enhances doxorubicin-induced apoptosis.

<p>NCI/ADR-RE cells were pretreated with MBO-asGCS (0–300 nM) and then exposed to doxorubicin (Dox. 5 µM) for 48 hr. a. Caspase-3/7 assay. RLU, relative luminescence units; *, p<0.005 compared with cells exposed to doxorubicin alone. b. Flow cytometry analysis. Apoptosis was quantitated by flow cytometry following propidium iodide staining (right panel). Left-hand bar graph is based on apoptotic cells detected on the sub-G0 phase. *, p<0.001 compared with cells exposed to doxorubicin alone. c. TUNEL staining for apoptosis. After pretreatment of MBO-asGCS or MBO-SC (50 nM), NCI/ADR-RE cells were exposed to doxorubicin (5 µM) for 48 hr. Apoptotic cells (TUNEL⁺) exhibit green fluorescence (x 200).</p

MBO-asGCS suppresses GCS expression in drug-resistant cancer cells.

<p>Drug-resistant and drug-sensitive cell lines (NCI/ADR-RE, MCF-7; EMT6/AR1, EMT6) were treated with MBO-asGCS (50 nM) for 48 hr. a. Quantitative RT-PCR. Isolated total RNA (100 ng/reaction) was synthesized to cDNA and analyzed by quantitative real-time PCR. b. Western blot. Total protein (50 µg/lane) was subjected to Western blot analysis; GCS expression levels are presented as the density ratio of GCS/GAPDH bands. c. Cellular ceramide glycosylation. After 24 hr MBO transfection, cells were incubated 500 µM NBD C₆-ceramide complexed to BSA. After 2 hr incubation, cellular sphingolipids were extracted and resolved by high-performance thin-layer chromatography and quantitated. ADR-RE, NCI/ADR-RE; *, p<0.001 compared with drug-sensitive cells; **, p<0.001 compared with corresponding vehicle control.</p

MBO-asGCS increases doxorubicin sensitivity in drug-resistant cancer cells.

<p>Cells (4,000/well) were plated in 96-well plates and pretreated with MBO-asGCS (50 nM). After 24 hr growth, cells were shifted to 5% FBS medium containing increasing concentrations of agents and grown for additional 72 hr. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay. a. Cell viability after C₆-ceramide treatment. ADR-RE, NCI/ADR-RE cells; *, p<0.01 compared with vehicle treatment. b. EC₅₀ values for C₆-ceramide. *, p<0.001 compared with vehicle treatments. c. Cell viability after doxorubicin treatment. d. EC₅₀ values for doxorubicin. *, p<0.001 compared with vehicle treatment.</p

Mixed-backbone oligonucleotide targeting human glucosylceramide synthase.

<p>a. Cellular uptake of MBO. After exposure of cancer cells to Cy3-labeled MBO-asGCS (50 nM) for the indicated periods, cellular Cy3-MBO-asGCS was measured at λ_excitation 550 nm and λ_emission 570 nm. b. Influence of MBOs on GCS expression. MBO-asGCS or MBO-SC was introduced into NCI/ADR-RE cells (ADR-RE) with Lipofectamine 2000 in Opti-EME I reduced-serum medium. After 48 hr growth, total RNA and protein were extracted. Total RNA (100 ng/reaction) was analyzed by RT-PCR. For Western blots, total protein (50 µg/lane) was subjected to 4–20% SDS-PAGE electrophoresis. Proteins were transferred to nitrocellulose and immunoblotted with GCS primary antibody (1∶500) and detected using ECL plus. GCS protein levels were presented as the ratios of the optical densities in GCS bands normalized against GAPDH.</p

Accumulations and effects of MBO-asGCS in normal tissues.

<p>a. MBO-asGCS accumulations. After 7 hr injection of Cy3-labeled MBO-asGCS (ip, 3 mice/each), tissues were removed and homogenized immediately. Fluorescence was measured and normalized against tissue weight. b. Apoptosis analysis. Mice were treated with combination of MBOs (1 mg/kg every three days, ip, for three times) and Dox (2 mg/kg, ip, for 48 hr; 3 mice/group). <i>Upper bar graph</i>, Caspase-3/7 activities in heart, lung, liver and kidney were assessed by using caspase-3/7 assay. RLU, relative luminescence units; *, p<0.05 compared with saline control. <i>Lower panel</i>, apoptotic cells in kidney were detected by using TUNEL staining.</p

MBO-asGCS suppresses GCS expression in tumors.

<p>Mice bearing NCI/ADR-RE tumors were treated with MBOs alone (1 mg/kg per 3 days, intratumoral injection) or in combination with doxorubicin (Dox, 2 mg/kg per week, intraperitoneal injection) for 38 days. Total RNA and proteins were immediately extracted from tumor homogenates. a. Quantitative RT-PCR for GCS. GCS mRNA levels were quantitated by using GCS standard and normalized against GAPDH. *, p>0.001 compared with saline or MBO-SC groups. b. Western blotting for GCS. Detergent-soluble protein (50 µg/lane) extracted from tumors (three per group) was incubated with anti-GCS or anti-GAPDH antibodies, following PAGE and transferring. c. Immunofluorescence microscopy. Tumor sections were stained with anti-GCS antibody (green). The nuclei were visualized by staining with DAPI (blue, x 200).</p