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. 2011 Mar 15;71(6):2276-85.
doi: 10.1158/0008-5472.CAN-10-3107. Epub 2011 Jan 28.

Suppression of Glucosylceramide Synthase Restores p53-dependent Apoptosis in Mutant p53 Cancer Cells

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Free PMC article

Suppression of Glucosylceramide Synthase Restores p53-dependent Apoptosis in Mutant p53 Cancer Cells

Yong-Yu Liu et al. Cancer Res. .
Free PMC article

Abstract

Tumor suppressor p53 plays an essential role in protecting cells from malignant transformation by inducing cell-cycle arrest and apoptosis. Mutant p53 that is detected in more than 50% of cases of cancers loses its role in suppression of tumors but gains in oncogenic function. Strategies to convert mutant p53 into wild-type p53 have been suggested for cancer prevention and treatment, but they face a variety of challenges. Here, we report an alternative approach that involves suppression of glucosylceramide synthase (GCS), an enzyme that glycosylates ceramide and blunts its proapoptotic activity in cancer cells. Human ovarian cancer cells expressing mutant p53 displayed resistance to apoptosis induced by DNA damage. We found that GCS silencing sensitized these mutant p53 cells to doxorubicin but did not affect the sensitivity of cells with wild-type p53. GCS silencing increased the levels of phosphorylated p53 and p53-responsive genes, including p21(Waf1/Cip1), Bax, and Puma, consistent with a redirection of the mutant p53 cells to apoptosis. Reactivated p53-dependent apoptosis was similarly verified in p53-mutant tumors where GCS was silenced. Inhibition of ceramide synthase with fumonisin B1 prevented p53 reactivation induced by GCS silencing, whereas addition of exogenous C6-ceramide reactivated p53 function in p53-mutant cells. Our findings indicate that restoring active ceramide to cells can resuscitate wild-type p53 function in p53-mutant cells, offering preclinical support for a novel type of mechanism-based therapy in the many human cancers harboring p53 mutations.

Figures

Figure 1
Figure 1
Silencing of GCS sensitized mutant p53 cancer cells to doxorubicin. A. Cell response to doxorubicin. NCI/ADR-RES cells were pretreated with MBO-asGCS for 7 days and exposed to doxorubicin for additional 72 hr. *, p<0.01 compared with vehicle control; **, p<0.001 compared with vehicle control. B. EC50 values for doxorubicin. Cells were pretreated with MBO-asGCS (50 nM) or vehicle (Lipofectamine 2000) for 7 days, and exposed to doxorubicin in 5% FBS medium for additional 72 hr. EC50 was calculated using Prism software after measurements. *, p<0.001 compared with vehicle control.
Figure 2
Figure 2
Disruption of ceramide glycosylation enhanced phosphorylated p53 and induces p21Waf1/Cip1 and Bax expressions in p53 mutant NCI/ADR-RES cells. A. GCS suppression in time-course. Cells were treated with MBO-asGCS (200 nM) and then exposed to doxorubicin (2.5 μM, 48 hr). Equal amount of detergent-soluble proteins (50 μg/lane) was resolved by 4–20% PAGE and immunoblotted with antibodies. *, p<0.001 compared with vehicle control; pp53, phosphorylated p53 (at Ser15 in DBD). B. GCS suppression in dose-dependent. Cells were treated with MBO-asGCS (7 days) or D-PDMP (48 hr) and then exposed to doxorubicin (2.5 μM, 48 hr). *, p<0.001 compared with vehicle control. C. Active pp53 and GCS in cells. A2780 cells were exposed to doxorubicin (Dox, 1 μM, 48 hr); NCI/ADR-RES cells were exposed to doxorubicin (Dox, 2.5 μM, 48 hr) following MBO-asGCS pretreatment (50 nM, 7 days). Merged fluorescence microphotographs (x200) was captured by confocal microscopy. Cells were recognized by anti-GCS (green) and anti-pp53 (red) antibodies with Alexa FluorR488- and Alexa FluorR555-conjugated goat antibodies. Nucleus was counterstained with DAPI (blue).
Figure 3
Figure 3
GCS suppression promoted mutant p53 cells to cell-cycle arrest and apoptosis. NCI/ADR-RES cells were exposed to doxorubicin (2.5 μM, for 48 hr) following MBO-asGCS pretreatments (50 nM, 7 days). Puma siRNA (siPuma, 100 nM) and its scrambled control (iSC, 100 nM) were introduced to cells in last 54 hr. A. Puma, a-Casp7 and c-PARP. Equal amount of proteins (50 μg/lane) was subjected to resolved and immunoblotted. B. Flow cytometry. PI stained cells were analyzed by flow cytometry. C. Cell division. D. Apoptotic cells. *, p<0.001 compared with vehicle control.
Figure 4
Figure 4
Silencing GCS by MBO-asGCS inhibited tumor growth through functional p53 restoration. A. Tumor growth curve. Athymic mice bearing NCI/ADR-RES tumors were treated with doxorubicin (Dox, 2 mg/kg/week) and combinations with MBO-asGCS (1 mg/kg/3-day) or MBO-SC (scrambled control) for 32 days. *, p<0.001 compared with the combination of MBO-SC and Dox. Data represents mean ± SEM (10 cases/group). B. Tumor proteins. Equal amount proteins extracted from tumors (100 μg/lane, 3 cases/group) were resolved by 4–20% PAGE and immunoblotted with antibodies, respectively. *, p<0.001 compared with MBO-SC. C. Immunostaining of pp53 and GCS (x 200). anti-GCS and anti-pp53 antibodies with Alexa FluorR488- and Alexa FluorR555-conjugated goat antibodies. Nucleus was counterstained with DAPI. D. TUNEL staining. Apoptotic cells (TUNEL+) exhibit green fluorescence (x200).
Figure 5
Figure 5
Ceramide restored p53 expression in mutant p53 cells. After pretreatments of MBO-asGCS (50 nM, 7 days), FB1 (25 μM, 48 hr) C6-ceramide (5 μM, 48 hr; C6-Cer,) and C6-dihydroceramide (5 μM, 48 hr; C6-diH-Cer), NCI/ADR-RES/asGCS cells were exposed to doxorubicin (2.5 μM, 48 hr). A. Western blotting. Equal amount proteins extracted from tumors (100 μg/lane) were resolved by 4–20% PAGE and immunoblotted with antibodies. *, p<0.001 compared with Vehicle. B. pp53 and ceramide in cells. Merged fluorescence microphotographs (x200) was captured by confocal microscopy. Green, cells were incubated with anti-pp53 (green) and anti-ceramide (red) following addition of Alexa FluorR488- and Alexa FluorR555-conjugated goat antibodies. Nucleus was counterstained with DAPI.

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